Abstract not available

The ability of low, clinically relevant levels of the chemotherapeutic drugs epirubicin and vinblastine to induce drug resistance was examined in the K562. U937, KG-la and HEL human leukaemia cell lines. Treatment with epirubicin and vinblastine induced the MDR phenotype and P-glycoprotein expression in K562 and U937 cells. However this treatment had no effect on drug resistance in the P-glycoprotein expressing KG-la and HEL cells. In the U937 cells, drug resistant cells were not only MDR but were also resistant to other drugs including cisplatinum and chlorambucil which are not normally associated with MDR. The drug resistant U937 sublines were also sensitised to doxorubicin, cisplatinum and chlorambucil by buthionine sulphoximine (BSO), suggesting that glutathione-related mechanisms also contributed to resistance in these sublines. The U937 sublines also had an increased DNA content and an increased ability to recover from DNA damage, as determined by cell cycle analysis, indicating that the broad cross-resistance exhibited by these cells was due to the co-existence of multiple resistance mechanisms. Drug treatment induced changes in expression of differentiation associated antigens in all four cell lines. Treatment with inducers of differentiation (TPA, sodium butyrate, granulocyte-macrophage colony-stimulating factor; GM-CSF). Treatment of K562 and K562/E15B cells with TPA induced megakaryocytic differentiation, with increases in drug resistance, and increased P-glycoprotein expression in the K562/E15B subline. TPA induced monocytic differentiation in the U937 cells but not the U937/EIS subline, with increased P-glycoprotein expression and function in the U937/E15 cells but not the U937 cells. Staurosporine, an inhibitor of PKC, inhibited differentiation in these cell lines, but did not inhibit increases in P-glycoprotein expression, suggesting drug resistance was not mediated by PKC. Sodium butyrate induced erythroid differentiation, and increased P-glycoprotein expression in the K562/E15B cells. However at a higher concentration (15 mM) this was not accompanied by increased drug resistance. Granulocyte monocyte colony stimulating factor (GM-CSF) did not induce differentiation in the K562 cells or K562/E15B subline, although the K562/E15B cells became more drug resistant after treatment with GM-CSF. Treatment with GM-CSF induced differentiation in the U937/E15 subline but did not change drug resistance in either the U937 cells or the U937/EI5 subline. Therefore the P-glycoprotein expressing K562/E15B and U937/E15 sublines were more responsive to inducers of differentiation than the parental cell lines. Induction of differentiation therefore induced increases in P-glycoprotein expression and drug resistance, suggesting that expression of P-glycoprotein or a multidrug resistance phenotype was associated with differentiation.

Lacosamide (LCM) is a new antiepileptic drug with a previously unknown mode of action. Using electrophysiological recording techniques in a range of in vitro preparations I have determined a mechanism of action of the new drug. In a 4-aminopyridine model of tonic-clonic seizures in rat visual cortex in vitro, LCM stereoselectively reduced maximal frequency and duration of tonic activity with EC[50�s] of 71 and 41 [mu]M respectively. LCM (100 [mu]M) significantly reduced excitability in whole cell patch clamped neurons producing non-selective reduction in the incidence of excitatory/inhibitory postsynaptic currents (EPSCs; LCM: 46.1 � 15.5 %, P <0.01, n = 4, IPSCs; LCM: 24.9 � 9.6 %, P <0.01, n = 4) and block of spontaneous action potentials (EC₅₀ 61 [mu]M). The inhibitory effects of LCM did not result from changes in passive membrane properties (including resting membrane potential or input resistance) as assessed by application of voltage ramps between -70 to +20 mV. LCM did not mimic the effects of diazepam as an allosteric modulator of GABA[A] receptor currents, nor did it inhibit evoked excitatory currents mediated by AMPA or NMDA receptors. Unlike phenytoin (DPH), carbamazepine (CBZ) or lamotrigine (LTG) that blocked sustained action potential firing evoked by brief depolarising steps (750 ms) or ramps (-70 to 20 mV, 90 mV.sec⁻�), LCM could weakly reduce the frequency of action potentials evoked by brief depolarisation suggesting a potential interaction with VGSCs. In accordance with this, the effect of LCM upon neurotransmission was negated in the presence of tetrodotoxin (200 nM, TTX). The frequency of miniature EPSCs was not altered by the drug (100 [mu]M). These results discounted some crucial potential anticonvulsant targets for LCM but implied a potential interaction with electrogenic VGSCs. When SRF duration was prolonged (10 s) LCM produced significant (P <0.01, n = 4-10, EC₅₀: 48 [mu]M) inhibition, but not within the first second of the burst EC₅₀: 640 [mu]M). Evoked TTX sensitive sodium currents in N1E-115 neuroblastoma cells were significantly reduced by LCM, CBZ, LTG and DPH when V[h]: -60 mV. Hyperpolarizing pulses (500 ms) to -100 mV could reverse block by CBZ, LTG and DPH but not LCM. The V₅₀ for steady state fast inactivation was more hyperpolarized by CBZ (-79.45 � 2.64 mV, n = 5, P < 0.001), LTG (-72.30 � 1.70 mV, n = 6, P <0.05) and DPH (-77.17 � 2.32 mV, n = 6, P <0.05) but not by LCM (-65.02 � 1.75 mV, n = 6, CONTROL: -65.84 � 0.86 mV). In contrast to CBZ, LCM did not slow recovery from fast inactivation or produce frequency dependent facilitation of block of a 3 s, 10 Hz pulse train. LCM (100 [mu]M) did produce a (V₅₀: CONTROL ~64 mV, LCM -57.47 � 4.53 mV, P <0.001, n = 4-8) hyperpolarizing shift in the voltage dependence of slow sodium channel inactivation and promoted channel entry into the slow inactivated state (P <0.001, n = 6) but did not alter the rate of recovery. I therefore conclude that LCM produces inhibition of epileptiform cellular activity, at least in part, via enhancement of voltage gated sodium channel slow inactivation and represents a molecule possessing a unique anticonvulsant mechanism of action.

Mucositis is the damage that occurs to the alimentary canal from anti - cancer therapies. It is caused by chemotherapy, radiotherapy and combination therapy and affects a large proportion of patients. Despite its prevalence, an effective anti - mucositis agent has yet to be developed that protects the whole tube, although the use of keratinocyte growth factor ( Amgen ' s Palifermin ) has recently been approved for the prevention of oral mucositis. It is important to understand mechanisms controlling mucositis so that treatment can be targeted appropriately. This thesis has investigated some of the key components identified as being involved in mucositis as well as identifying new genes which contribute to chemotherapy - induced intestinal injury. The research chapters investigated : 1 ) Gene expression of the apoptosis - regulating Bcl - 2 family, p53 and caspase - 3, and the changes which occur in the intestine following chemotherapy treatment for cancer. 2 ) The effect of different chemotherapeutic agents on intestinal cells in vitro and the role p53 plays. 3 ) The mucositis caused by single dose irinotecan in the rat with breast cancer and the role of p53 in induction of intestinal damage. 4 ) The early gene changes that occur in the small intestine of the rat with breast cancer following irinotecan treatment. Firstly, to investigate the difference in susceptibility to damage between the small and large intestine, the protein expression of 8 members of the Bcl - 2 family ( 4 pro - apoptotic ; Bax, Bak, Bid, Bim and 4 anti - apoptotic ; Bcl - 2, Bcl - xL, Bcl - w, Mcl - 1 ) was quantified in jejunal and colonic sections taken from rats inoculated with breast cancer. It was found that there was significantly higher expression of the pro - apoptotic proteins, Bax, Bak, Bim and Bid, in the crypts of the jejunum compared to the colon. Furthermore, expression of the anti - apoptotic proteins, Bcl - 2, Bcl - xL and Bcl - w, was significantly lower in jejunal crypts compared to colonic crypts. Mcl - 1 expression was similar in both regions. Thus, the small intestine is an environment balanced to favour apoptosis through specific Bcl - 2 family protein expression profiles. The Bcl - 2 family regulates apoptosis in response to a variety of chemotherapy agents. However, it is unknown how Bcl - 2 family gene expression changes along with other apoptogenic factors following cytotoxic therapy in the normal intestine. To investigate this, sections of rat jejunum treated with methotrexate and duodenal biopsies from chemotherapy patients treated with various regimens for cancer were subjected to quantitative immunohistochemistry to detect Bcl - 2 family proteins, p53 and caspase - 3. Treatment caused expression of p53 and caspase - 3 to increase within the crypts and follow a similar pattern to apoptosis levels. Pro - apoptotic Bcl - 2 family members, Bax and Bak, were increased, while the anti - apoptotic protein, Mcl - 1, was significantly reduced. A significant increase in mRNA expression for Bax and Bak was noticed at 6 h, without a concurrent decrease in Mcl - 1. Thus, Bcl - 2 family genes were altered in the small intestine in both humans and rats, and this was irrespective of chemotherapy agent or regimen used. The best characterised changes which occur during chemotherapy - induced damage in the intestine are in the epithelial layer, although it is thought that pan #45 mucosal alterations are involved. Two intestinal cell lines were chosen to investigate changes in apoptosis, proliferation and protein expression following cytotoxic treatment with various chemotherapeutic agents. These were the rat IEC - 6 and human FHs 74 cell lines, which represent untransformed epithelial cells. The human breast carcinoma cell line, MCF - 7, was also used as a positive control. Intestinal cells were resistant to the occurrence of methotrexate toxicities within 24 h of treatment, modestly affected by irinotecan and extremely sensitive to doxorubicin. Doxorubicin caused a marked increase in p53 and p21 expression, which for irinotecan was less pronounced. The effect of cytotoxic treatment on Bcl - 2 family expression in intestinal cells varied, however the pro - apoptotic proteins, Bax and Bak, were generally upregulated following doxorubicin. Temporary inhibition of p53 using pifithrin alpha resulted in a significant improvement in cell survival in cancerous cell only and did not alter Bcl - 2 family expression. It was concluded that cultured epithelial cells exhibit varying sensitivities to different chemotherapeutic agents which is dependent on induction of p53 gene expression. The topoisomerase I inhibitor, irinotecan, is a chemotherapeutic agent commonly used in the treatment of colorectal cancer. It often induces severe mucositis with the most common symptom being diarrhoea. Previous research has shown that irinotecan damages the small and large bowel equally, which is unusual. This is characterised by an increase in apoptosis and a reduction in proliferation within epithelial crypts, an increase in inflammatory cell infiltrate in the lamina propria and excess mucin production. These investigations used two sequential doses of irinotecan. The early effect of a single dose of irinotecan on the intestine have yet to be studied. Thus the primary aim of this experiment was to examine in detail the changes caused by irinotecan at 6 and 48 h in the rat. A secondary aim was to investigate the role of p53 on induction of apoptosis and cell cycle arrest within intestinal crypts and the effect of temporary inhibition of the protein. Single dose irinotecan caused a decrease in body and small intestinal weight by 48 h after treatment. This was accompanied by crypt and villous degeneration, increased apoptosis and reduced proliferation within crypt epithelium as well as inflammatory infiltrate throughout lamina propria. An increase in Bax expression was seen at 6 h, however p53 protein levels remained relatively low until 48 h. Rats also treated with pifithrin alpha to inhibit p53 and had a significantly lower peak in apoptosis in the colon at 6 h, however did not show improvements in any other parameters tested. It was concluded that irinotecaninduced damage in the rat intestine is primarily p53 - independent, and that pifithrin alpha acts to inhibit apoptosis in the large intestine via a p53 - independent pathway. A study was designed to investigate the early genome - wide changes which occur following irinotecan treatment in the rat small intestine. Microarray analysis found that regulation of many genes was altered at 6 h following dual dose irinotecan. These genes were involved in apoptosis, cell cycle regulation, immune function, calcium homeostasis and protein turnover. Multiple genes from the MAP kinase pathway were also activated by irinotecan. The cystine protease, caspase - 1 was upregulated and was chosen for further investigations due to its role in apoptosis and inflammation. Real time PCR analysis confirmed the increase in gene expression at 6 h and also showed a return to baseline levels by 24 h which was followed by another modest increase at 48 h. It was concluded that irinotecan induces a wide range of gene changes within the intestine and that apoptosis and inflammatory damage pathways are activated during treatment. This thesis described key molecules in apoptosis and their role in induction of chemotherapy - induced intestinal mucositis. It has provided evidence of the importance of apoptosis in mucosal injury and also highlighted areas requiring further research. Results presented herein show that the Bcl - 2 family is involved in intestinal damage following many chemotherapy agents, whereas p53 is agent - specific. It has also shown that irinotecan causes intestinal damage via a mainly p53 - independent manner in the rat. It can be concluded that gastrointestinal mucositis is complex and activates multiple pathways to induce damage. Findings from this thesis will aid targeting of new anti - mucotoxic agents. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2006.

Copy of author's previously published article inserted. / Bibliography: leaves 210-234. / xiii, 235 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the effect of chemotherapy on the mucosa of the small intestine and the prevalence, duration and severity of mucositis, both in humans and in rats. / Thesis (M.D.)--University of Adelaide, Depts. of Gastroenterology and Haematology/Oncology, 1998

Articles and serials related to the thesis research bound in appendix / Includes bibliographical references / v, 217 leaves, [2] leaves of plates : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (M.D.)--University of Adelaide, Faculty of Medicine, 1984

Copy of author's previously published article inserted. / Bibliography: leaves 210-234. / xiii, 235 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the effect of chemotherapy on the mucosa of the small intestine and the prevalence, duration and severity of mucositis, both in humans and in rats. / Thesis (M.D.)--University of Adelaide, Depts. of Gastroenterology and Haematology/Oncology, 1998

<p>Illamående och kräkning är förekommande biverkningar hos patienter med cancer som behandlas med cytostatika och är därmed tillstånd som sjuksköterskan kommer i kontakt med. Om patientens illamående och kräkning inte förebyggs kan det leda till konsekvenser som att patienten blir dehydrerad, tappar aptiten, blir undernärd och försvårar skadad vävnad att återhämta sig från cytostatikabehandling. Trots att de flesta patienter får antiemetika lyckas inte deras illamående och kräkning förebyggas. Syftet var att belysa komplementära behandlingsmetoder vid cytostatikarelaterat illamående och kräkning. Studien är utförd som en litteraturstudie och baserad på 14 vetenskapliga studier. Resultatet visar att komplementära behandlingsmetoder har ett visst vetenskapligt stöd och är ett bra komplement till antiemetika. Akupressur är den behandlingsmetod som är effektivast för att förebygga cytostatikarelaterat illamående och kräkning. Även akustimulation, elektroakupunktur och avslappning minskar patienternas illamående och kräkning. Mer forskning behövs för att ge mer styrka åt de olika komplementära behandlingsmetoder, eftersom sjuksköterskans arbete måste utföras utifrån vetenskap och beprövad erfarenhet. Genom att sjuksköterskan får ökad kunskap om hur cytostatikarelaterat illamående och kräkning förebyggs kan patientens lidande minska.</p>

<p>J1 (L-melphalanyl-L-<i>p</i>-fluorophenylalanyl ethyl ester) is a dipeptide derivative of the alkylating agent melphalan with increased cytotoxicity. In this thesis the preclinical pharmacology of J1 has been characterized.</p><p>Our results show that J1 rapidly enters the cells, where melphalan is released by hydrolysis. The maximum concentration (C_max) of melphalan was detected 15 min after exposure to J1 in human cancer cell lines. In comparison, melphalan exposure resulted in a 10-fold lower C_max that was shifted to later time points. J1 induced more DNA damage and apoptosis than melphalan. The cytotoxic activity and release of melphalan from J1 were inhibited by preincubating cells with the aminopeptidase inhibitor bestatin. In accordance with these results, we showed that J1 is a substrate for aminopeptidase N (APN), which may result in increased tumor selectivity.</p><p>J1 effectively inhibited cell growth in a set of neuroblastoma cell lines.<i> </i>Athymic mice carrying neuroblastoma xenografts were treated either with equimolar doses of melphalan or J1. J1 inhibited the tumor growth more effectively than melphalan and the untreated control, and was associated with higher caspase-3 activation, fewer proliferating tumor cells and decreased mean vascular density.</p><p>J1 and melphalan showed similar activity profiles when tested in 176 primary tumor cell cultures from patients, but J1 exhibited 50- to 100-fold higher potency. The difference was greater in some diagnoses (e.g. breast cancer, NHL and AML), and was exceptionally large in some breast cancer samples with aggressive phenotypes. A combination screening of J1 and standard chemotherapeutics yielded mostly additive interactions, except for etoposide which induced synergy in all tested cell lines.</p><p>In conclusion, the melphalan prodrug J1 is effectively transported into the cells, where aminopeptidases (for example APN) catalyze the formation of melphalan. J1 shows promising preclinical potential in the diagnoses neuroblastoma and breast cancer.</p>

A unique tumor targeted method, which may be able to deliver a molecule to the surface of a tumor cell using the pH gradient between hypoxic tumor cells and normal tissue has recently been developed. Since solid tumors have been found to have a lower extra cellular pH compared to normal tissue (6.5 to 6.9 for tumors verses an average 7.4 for normal tissue), the pH gradient is used as a source of power to activate a strategically designed "molecular engine" capable of delivering a diagnostic or therapeutic agent to tumor cells. To test this hypothesis, a 22- sequence amino acid, which reorganizes to alpha helical form at pH 6.9 causing the molecule to become lipophilic and embed into the plasma membrane of nearby cells was synthesized. The molecule was then attached to 99mTc via a MAG-3 chelating molecule. In-vivo nuclear imaging was performed and showed apparent significant uptake in primary tumors as well as lung and liver in Lewis lung cell model C57blk-J6 mice with confirmed primary tumors at the base of the tail or lungs. This study shows significant promise for early diagnosis and treatment of cancer on a molecular level. / Graduation date: 2006