Small cell lung cancer (SCLC) is characterized by prompt response to chemotherapy and radiotherapy but relapsing with drug resistance and distant metastasis, leading to poor overall prognosis. New anticancer agents and regimens are drastically needed for SCLC treatment. Arsenic trioxide (ATO), a traditional Chinese medicine used as a poison for thousands of years, has been tested in many hematological and solid cancers both in vitro and in vivo, with promising effects. In order to establish the scientific ground for future clinical application of ATO in SCLC, this study aimed to investigate the anticancer effect and mechanism of ATO in SCLC using in vitro and in vivo models, either as a single agent or in combination with standard chemotherapy. In addition, an ATO-acquired resistant cell line (H841-AR) derived from SCLC cell line H841 was used to explore potential mechanisms of ATO resistance and cross-resistance to other chemotherapeutic drugs. In the first part of this study, ATO was shown to exert cytotoxic effect in all of the chosen SCLC cell lines. Various cellular mechanisms were triggered upon ATO exposure: redox status disturbances (hydrogen peroxide (H2O2) generation, glutathione (GSH) depletion and thioredoxin 1 (Trx1) down-regulation), mitochondrial membrane depolarization (MMD), DNA damage, apoptosis and necroptosis. In concert with this, Bcl-2 was down-regulated accompanied by MMD, release of AIF and SMAC, DNA degradation, XIAP inhibition and caspases activation. Adoption of N-acetyl-L-cysteine (NAC) and buthionine sulfoximine (BSO) demonstrated GSH depletion and reactive oxygen species (ROS) generation played the pivotal role to mediate cytotoxic effect of ATO in SCLC. In the second part of this study, when combined with chemotherapeutic agents, ATO displayed synergistic and antagonistic interaction with cisplatin and etoposide respectively in SCLC cell line models. The beneficial combination of ATO and cisplatin was also substantiated by tumor xenograft models. Augmented GSH depletion and suppressed drug efflux mechanism were found to explain the synergistic effects. In the last part of this study, H841-AR was generated as an acquired multi-drug resistant (ATO, cisplatin and etoposide) cell line to investigate the potential resistance mechanisms and possible future drug combinations. Comparing H841-AR cells with parental H841 cells using cDNA microarray, a long list of genes was altered in ATO-resistant cells. At least 20 up-regulated and 45 down-regulated genes were short-listed as candidates with a cut-off at 5-fold change. Interestingly, qPCR data has shown that 5 selected up-regulated genes in H841-AR cells were also highly expressed in DMS79 cells with intrinsic ATO resistance compared to the relatively sensitive cell lines, indicating that these genes might be associated with ATO resistance in SCLC. In summary, ATO was shown to be an active anticancer agent in SCLC, either alone or in combination with cisplatin. The major mechanisms of action of ATO and its synergism with cisplatin in SCLC were elucidated. Genetic data derived from an acquired resistant (to ATO, cisplatin and etoposide) SCLC cell line may help to uncover the mechanisms of resistance to ATO, allowing possible future drug combinations. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Lung cancer is the leading cause of cancer deaths worldwide. A large part of this high mortality rate is due to the onset of metastatic disease prior to diagnosis. Advances in treatment for metastatic disease may be achieved by understanding more about the identity of metastatic tumor cells and the mechanisms those cells employ to spread throughout the body. This thesis examined the relationship between cells capable of tumor propagation upon serial transplantation (tumor-propagating cells, or TPCs) and those with metastatic potential.

Macrophages are cells of the innate immune system that can be found in large quantities in cancer tumors and affect cancer progression by regulating growth and invasiveness of cancer cells. There are two main phenotypes of macrophages denoted M1 and M2. In this thesis, the M1 and M2 phenotype of human macrophages were characterized, and effects of the macrophages on the growth and invasiveness of colon and lung cancer cells were studied. Macrophages of the M1 phenotype, but not the M2 phenotype, inhibited growth of both colon and lung cancer cells, and the inhibition for some of the cancer cell lines was induced by cell cycle arrest in the G1/G0 and/or G2/M cell cycle phases. In the colon cancer cell line, the macrophage induced cell cycle arrest was found to attenuate the cytotoxic effect of the chemotherapeutic drug 5-FU. Macrophages were also shown to express high levels of proteases (matrix metalloproteinase-2 and 9) and high levels of proteins of the urokinase-type plasminogen activator (uPA) system, in comparison to the lung cancer cell lines studied. Expression of these has been found to predict poor outcome in lung cancer, and the results suggest macrophages to be important contributors of these in lung tumors. Furthermore, the M1 phenotype was found to express higher levels of the uPA receptor than the M2 phenotype. Prostaglandin E2 (PGE2) is a potent inflammatory molecule expressed by e.g. macrophages and monocytes, and inhibition of its expression has been shown to reduce the risk of colon cancer. Green tea and black tea was found to be potent inhibitors of PGE2 formation in human monocytes, and the inhibitory effects of green tea was likely due to its content of the polyphenol epigallocatechin gallate. Rooibos tea also inhibited PGE2 formation, but was less potent than green and black tea. The primary mechanism for the inhibition was via inhibition of expression of enzymes in the PGE2 formation pathway, and primarily microsomal prostaglandin synthase-1. / Macrophages are cells of the immune system often found in large numbers in cancer tumors. They affect multiple aspects of cancer progression, including growth and spread of cancer cells, and the efficacy of treatments. There are two major macrophage phenotypes denoted M1 and M2, that have mainly pro- and anti-inflammatory properties, respectively. In this thesis, M1 and M2 macrophages were characterized and effects of them on different aspects of cancer progression were studied using culture of colon, and lung cancer cells. The M1 phenotype inhibited proliferation of cancer cells from both colon and lung. The growth inhibition was for some cell lines accompanied by cell cycle arrest. The macrophage induced cell cycle arrest was found to protect colon cancer cells from the cytostatic drug 5-fluorouracil. Prostaglandin E2 (PGE2) contributes to colon cancer development and treatment of monocytes with tea extracts inhibited PGE2 formation via inhibition of expression of microsomal prostaglandin E synthase-1. Proteases can degrade the extracellular matrix of a tumor to facilitate cancer cell invasion and metastasis. The M1 and M2 phenotypes of macrophages expressed several protease activity related genes to a greater extent than lung cancer cells, and M1 more so than the M2 phenotype.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Είναι γνωστό ότι οι μεταγγίσεις ομόλογου αίματος ασκούν ανοσοκατασταλτική δράση, ευνοόντας την υποτροπή των καρκινικών όγκων και των μεταστάσεων. Επιπλέον η περιεγχειρητική αναιμία θεωρείται ανεξάρτητος προγνωστικός παράγων σε ασθενείς που υποβάλλονται σε ογκολογικές επεμβάσεις. Οι μείζονες θωρακικές επεμβάσεις, όπως αυτές για καρκίνο του πνεύμονα συνοδεύονται από αυξημένες απώλειες αίματος και ανάγκες για μετάγγιση οδηγώντας σε επακόλουθη αύξηση της νοσηρότητας και θνησιμότητας. Παράλληλα η ευεργετική επίδραση της απροτινίνης είναι ευρέως γνωστή όσον αφορά τις Καρδιοχειρουργικές επεμβάσεις. Σκοπό των μελετών μας αποτέλεσε, από τη μία η πιθανή επίδραση των μεταγγίσεων ομολόγου αίματος και της προεγχειρητικής αναιμίας στην απώτερη επιβίωση των ασθενών που υπεβλήθησαν σε εκτομή μη μικροκυτταρικού καρκίνου του πνεύμονα και από την άλλη η επίδραση της απροτινίνης στις ανάγκες για μετάγγιση και την αιμορραγική διάθεση, χορηγούμενη σε πολύ χαμηλή δόση σε μείζωνες πνευμονικές εκτομές. Κατά την πρώτη μας μελέτη, 331 ασθενείς (άνδρες/γυναίκες=295/36, μέσης ηλικίας 64 ± 9 έτη), οι οποίοι υπεβλήθησαν σε ριζική εκτομή μη μικροκυτταρικού καρκίνου πνεύμονα, εξετάσθηκαν προοπτικά. Ο μέσος χρόνος μετεγχειρητικής παρακολούθησης ήταν 27,2 μήνες. Η συνολική επιβίωση των ασθενών εξετάσθηκε συγκριτικά με την χορήγηση μεταγγίσεων ομολόγου αίματος και την περιεγχερητική αναιμία. Οι παράμετροι αυτοί εξετάσθηκαν αρχικά για τον συνολικό πληθυσμό ασθενών και εν συνεχεία ξεχωριστά για τους ασθενείς σταδίου I. Οι ασθενείς ταξινομήθηκαν ανάλογα με την χορήγηση περιεχειρητικής μετάγγισης αίματος στην Ομάδα Α (μεταγγιζόμενοι) και Ομάδα Β (μη μεταγγιζόμενοι) και ανάλογα με το επίπεδο της προεγχειρητικής αιμοσφαιρίνης (Hb) στην Ομάδα 1 (Hb<12 g/dl) και Ομάδα 2 (Hb≥12 g/dl) αντίστοιχα. Κατά τη δέυτερη μας μελέτη, μια υποομάδα 59 ασθενών (μέσης ηλικίας 58 ± 13.25 έτη) οι οποίοι υπεβλήθησαν σε μείζονα θωρακοχειρουργική επέμβαση ταξινομήθηκαν στην ομάδα ελέγχου (Ομάδα Α) και στην ομάδα απροτινίνης (Ομάδα Β). Η δεύτερη ομάδα (n=29) έλαβε κατά την εισαγωγή της αναισθησίας διεγχειρητικά 500.000 I.U απροτινίνης, ακολουθούμενη από μία δεύτερη ισόποση δόση αμέσως μετά το κλείσιμο της θωρακοτομής. Τα αποτελέσματά μας για την πρώτη μελέτη κατέγραψαν ποσοστό μετάγγισης 25,7%. Η μονοπαραγοντική ανάλυση για το σύνολο του πλυθησμού ανέδειξε συνολική επιβίωση μικρότερη στην Ομάδα Α (μεταγγιζόμενοι) (μέση επιβίωση 33.6 μήνες, 5-ετής επιβίωση 25.1%) σε σύγκριση με την Ομάδα B (μέση επιβίωση 48.0 μήνες, 5-ετής επιβίωση 37.3%) (p=0.001). Παρατηρήσαμε επίσης ότι οι ασθενείς με προεγχειρητική τιμή Hb<12 g/dl (Ομάδα 1) (μέση επιβίωση 33.0 μήνες, 5-ετής επιβίωση 21.3%), παρουσίασαν μικρότερη επβίωση συγκρινόμενοι με την Ομάδα 2 (μέση επιβίωση 49.3 μήνες, 5-ετής επιβίωση 40%) (p=0.002). Η πολυπαραγοντική ανάλυση του συνόλου των ασθενών ανέδειξε ότι η προεγχειρητική αναιμία αποτελεί ανεξάρτητο προγνωστικό παράγοντα για την επιβίωση, ενώ η μετάγγιση με μονάδες ομόλογου αίματος όχι. Παρατηρήσαμε επίσης κατά την πολυπαραγοντική ανάλυση ότι στους ασθενείς του σταδίου I, οι μεταγγίσεις ομόλογου αίματος αποτέλεσαν ανεξάρτητο προγνωστικό παράγοντα για την απώτερη επιβίωση, γεγονός που δεν επιβεβαιώθηκε για την προεγχειρητική αναιμία. Στην δεύτερη μελέτη μας, η μέση παροχή αιματηρού περιεχομένου που κατεγράφη από τους σωλήνες θώρακος κατά την 1η και 2η μετεγχειρητική ημέρα στην Ομάδα Β (απροτινίνης) ήταν σημαντικά ελαττωμένη (412.6±199.2 έναντι 764.3±213.9 ml, p<0.000 και 248.3±178.5 έναντι 455.0±274.6, p<0.001 αντίστοιχα). Παρομοίως, οι ανάγκες μετάγγισης με φρέσκο κατεψυγμένο πλάσμα ήταν λιγότερες στην Ομάδα της απροτινίνης. Επιπλέον, τόσο ο διεγχειρητικός χρόνος, όσο και η διάρκεια παραμονής στο νοσοκομείο ήταν χαμηλότερα υπέρ της Ομάδας Β, χωρίς όμως να αγγίζουν την στατιστική σημαντικότητα (84.6±35.2 έναντι 101.2±52.45 λεπτών και 5.8±1.6 έναντι 7.2±3.6 ημερών αντίστοιχα) (p<0.064). το συνολικό ποσοστό μετάγγισης δεν δέφερε σημαντικά ανάμεσα στις δύο ομάδες. Δεν παρατηρήθηκαν ανεπιθύμητες ενέργειες κατά τη χορήγηση της ουσίας. Τα συμπεράσματά μας συνιστούν ότι οι μεταγγίσεις ομολόγου αίματος επηρεάζουν αρνητικά την απώτερη επιβίωση των ασθενών με πρώιμο μη μικροκυτταρικό καρκίνο (σταδίου I) του πνεύμονα, χωρίς να παρατηρείται όμως η ίδια επίδραση στην επιβίωση αυτών με χειρουργικά εξαιρέσιμη πιο προχωρημένη νόσο. Τα ευρήματα αυτά καταδεικνύουν ότι οι μεταγγίσεις ομολόγου αίματος πιθανώς να ασκούν ανοσοτροποποιητική δράση στα πρώιμα στάδια της νόσου, ενώ για τα πιο προχωρημένα το φιανόμενο αυτό δεν είναι προφανές. Επιπλέον, η χορήγηση της απροτινίνης σε πολύ χαμηλή δόση, συνοδεύεται από ελάττωση των μετεγχειρητικών απωλειών αίματος και αναγκών για μετάγγιση των παραγώγων του αίματος. Με βάση τα δεδομένα αυτά θεωρούμε την χορήγηση της απροτινίνης ως ασφαλή και ευεργετική στις μείζονες θωρακοχειρουργικές επεμβάσεις. / It has been postulated that transfusions have immunosuppressive effects that promote tumor growth and metastasis. Moreover perioperative anaemia is considered an independent prognostic factor on outcome in patients operated for malignancy. Major thoracic operations (such as lung cancer resections) are associated with increased blood losses and transfusion requirements leading to increased mortality and morbidity. In addition, the blood saving effect of aprotinin has been well documented in cardiac surgery. The aims of our studies were to evaluate, on the one hand the possible influence of red blood cell (RBC) transfusions and perioperative anaemia on survival in patients operated for non-small cell lung carcinoma (NSCLC); and on the other hand, we have tested the influence of aprotinin using an ultra-low dose drug regime on major pulmonary operations concerning bleeding diathesis and need for transfusion. In our first study, 331 consecutive patients, male/female=295/36, (mean age 64±9 years), who underwent radical surgery for NSCLC were prospectively enrolled in this cohort and followed up for a mean of 27.2 months. The overall survival of patients was analyzed in relation to RBC transfusions and perioperative anemia. These parameters were analyzed in the whole cohort of patients and separately for stage I patients. Patients were divided according to perioperative transfusion, into Group A (transfused) and Group B (non-transfused) and according to the preoperative haemoglobin (Hb) level into Group 1 (Hb<12g/dl) and Group 2 (Hb≥12gr/dl) respectively. Furthermore in our second study, a subgroup of 59 patients, of mean age 58±13.25 years (mean±SD) undergoing general major thoracic procedures were randomized into placebo (Group A) and treatment–aprotinin group (Group B). The group B (n=29) received 500.000 IU of aprotinin after induction to anesthesia and a repeat dose immediately after chest closure. In our fisrt study, the overall transfusion rate was 25.7%. Univariate analysis showed that in the whole cohort of patients overall survival was significantly shorter in Group A (mean 33.6 months, 5-year survival 25.1%) compared to Group B (mean 48.0 months, 5-year survival 37.3%) (p=0.001). It also showed that patients with preoperative Hb level<12g/dl (Group 1), (mean of 33.0 months, 5-year survival 21.3%) had shorter survival compared to Group 2 patients (mean 49.3 months and 5-year survival 40.0%) respectively (p=0.002). Multivariate analysis in the whole cohort of patients showed that preoperative anemia was an independent risk factor for survival while RBC transfusion was not. In particular for stage I patients, it was shown that RBC transfusion was an independent prognostic factor for long-term survival as detected by multivariate analysis (p=0.043), while anemia was not. In our second study, the two groups were similar in terms of age, gender, diagnosis, pathology, comorbidity and operations performed. The mean drainage of the first and second postoperative day in group B was significantly reduced (412.6±199.2 vs. 764.3±213.9 ml, p<0.000, and 248.3±178.5 vs. 455.0±274.6, p<0.001). Similarly, the need for fresh frozen plasma transfusion was lower in group B, p<0.035. Both the operation time and the hospital stay were also less for group B but without reaching statistical significance (84.6±35.2 vs 101.2±52.45 min. and 5.8±1.6 vs 7.2±3.6 days respectively, p<0.064). The overall transfusion rate did not differ significantly. No side effects of aprotinin were noted Our conclusions suggested that RBC transfusions affect adversely the survival of stage I NSCLC patients, while do not exert any effect on survival of patients with surgically resectable more advanced disease, where preoperative anemia is an independent negative prognostic factor. These findings indicate that RBC transfusion might exert an immunomodulatory effect on patients with early disease while in more advanced stages this effect is not apparent. Additionally perioperative ultra-low dose aprotinin administration was associated with a reduction of total blood losses and blood product requirements. We therefore consider the use of aprotinin safe and effective in major thoracic surgery.

No description available.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumour which secretes ACTH and other related peptides. Contrary to normal production by the pituitary, ACTH production is not inhibited by glucocorticoids (Gcs) in SCLC. This insensitivity to Gc action can be attributed to impaired Gc receptor (GR) expression in these cells. Over-expression of the GR induces apoptosis both in vitro and in vivo. Evasion of GR signalling thus confers a significant survival advantage to SCLC cells. Re-expression of endogenous GR in SCLC cells may provoke the same effect. Many tumours silence the expression of tumour suppresser genes by epigenetic mechanisms. Recent evidence suggests that the GR in SCLC cells is epigenetically silenced by hypermethylation of its promoter. The overall aim of this study was to determine whether endogenous GR re-expression induces apoptosis of SCLC cells. The DMS 79 SCLC cell line, and the control HEK and non-SCLC A549 cell lines were treated with the DNA methyltransferase inhibitor (DNMTi), 5-aza-2′-deoxycytidine (5-aza), to determine whether treatment with 5-aza results in re-expression of endogenous GR. Conflicting results were thought to result from the use of possibly degraded 5-aza. However, a quantitative real-time PCR analysis using newly purchased, freshly prepared 5-aza indicated that 5-aza treatment up-regulated GR mRNA expression in the DMS 79 cells (p<0.0005). No significant changes in GR expression were seen in the HEK and/or A549 cells, suggesting that the GR in these cell lines is not methylated. Contrary to expectations and possibly due to the use of degraded stock, Western blot analysis revealed that 5-aza had no effect on GR protein expression in DMS 79 cells, yet affected GR protein expression in HEK and A549 cells (p=0.003 and p=0.042, respectively). Cell viability assays indicated that treatment with varying concentrations of 5-aza had no effect on the viability of DMS 79 and A549 cells, but had a minimal effect on HEK cell (p<0.0005) viability. These data reinforce the hypothesis that stock 5-aza had degraded as 5-aza is known to exert cytotoxic effects at higher concentrations. Using newly purchased, freshly prepared 5-aza, flow cytometry and/or microscopy were performed to establish whether endogenous GR re-expression was sufficient to kill the SCLC cells by apoptosis. FITC Annexin V staining and nuclear morphology showed that significant proportions of the 1 μM (p=0.010 and p=0.027) and 5 μM (p=0.002 and p=0.018) 5-aza treated DMS 79 cells were apoptosing, with little apoptosis seen in HEK cells. 5-Aza induced negligible HEK cell death, as determined by microscopic analyses. The effect of dexamethasone (Dex; a synthetic Gc) on HEK and DMS 79 cells was examined to determine whether Gc treatment could enhance apoptosis. Treatment with Dex alone, and in combination with 5-aza, resulted in significant HEK cell death (p=0.046 and p=0.005 respectively), but not apoptosis. This was unexpected as HEK cells express very little unmethylated GR, and may be due to excessive drug exposure or combined drug toxicity. The same effect was observed with DMS 79 cells (p=0.003 and p<0.0005 respectively), with 5-aza appearing to enhance cell death induced by Dex. No effects on apoptosis were seen confirming earlier reports that GR-mediated apoptosis is ligand-independent. As 5-aza does not selectively demethylate the GR, cells were exposed to the GR antagonist, RU486, to establish whether apoptosis associated with 5-aza treatment is specifically due to demethylation and subsequent expression of the GR. Treatment with RU486 in conjunction with 5-aza induced cell death (p=0.014), but not apoptosis, of HEK cells. Again, this may have been due to excessive drug exposure or combined drug toxicity. Flow cytometric data showed that DMS 79 cell death was induced by both RU486 (p=0.004), and RU486 in combination with 5-aza (p=0.003). Furthermore, although not significant, RU486 treatment appeared to inhibit apoptosis induced by 5-aza in the DMS 79 cells. The data suggest that re-expression of the GR may be responsible for apoptotic induction. Our findings, although not significant, hint that endogenous re-expression of the GR leads to apoptosis. Unlike mutations, epigenetic marks are reversible and clinical trials with DNMTis have shown promising results. The identification of a novel endogenous mechanism that specifically induces apoptosis of SCLC cells offers great promise for the development of targeted therapeutics for the treatment of this deadly disease. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.

We explore both robust biologically guided intensity-modulated radiation therapy (BG-IMRT) and pattern recognition to identify responders to cancer treatment for lung cancer. Heterogeneous dose prescriptions that are derived from biological images are subject to uncertainty, due to potential noise in the image. We develop a robust optimization model to design BG-IMRT plans that are de-sensitized to uncertainty. Computational results show improvements in tumor control probability and deviation from prescription dose compared to a non-robust model, while maintaining tissue dose below toxicity levels. We applied machine learning algorithms to 4D gated positron emission tomography/computed tomography (PET/CT) scans. We identified classifiers which could outperform a naive classifier. Our work shows the potential of using machine learning algorithms to predict patient response. This could hopefully lead to more adaptive treatment plans, where the clinician would adapt the treatment based on the prediction provided at certain time intervals in the treatment.

We explore both robust biologically guided intensity-modulated radiation therapy (BG-IMRT) and pattern recognition to identify responders to cancer treatment for lung cancer. Heterogeneous dose prescriptions that are derived from biological images are subject to uncertainty, due to potential noise in the image. We develop a robust optimization model to design BG-IMRT plans that are de-sensitized to uncertainty. Computational results show improvements in tumor control probability and deviation from prescription dose compared to a non-robust model, while maintaining tissue dose below toxicity levels. We applied machine learning algorithms to 4D gated positron emission tomography/computed tomography (PET/CT) scans. We identified classifiers which could outperform a naive classifier. Our work shows the potential of using machine learning algorithms to predict patient response. This could hopefully lead to more adaptive treatment plans, where the clinician would adapt the treatment based on the prediction provided at certain time intervals in the treatment.