Search

The chemopreventive agent indole-3-carbinol (I3C) is found in cruciferous vegetables such as broccoli, cabbage and Brussels sprouts. Clinical trials have shown I3C to favourably alter urinary 2-OH:16a estrone ratios, which may prove beneficial for decreased risk of ER+ve breast cancer. However, molecular mechanisms for potential chemopreventive effects in ER-ve breast cancer have yet to be elucidated. I3C inhibited proliferation of the immortalised HBL 100, and tumour-derived MDA MB468, T47D and MCF7 breast cell lines. Approximate IC50s were 122mNm 33mM, 73mM and 67mM respectively, with MDA MB468 cells exhibiting four-fold greater inhibition than HBL 100 cells. I3C caused significant induction of apoptosis in the MDA MB468 cells only, possibly explaining this difference in sensitivity. This was not due to immediate effects upon members of the Bcl-2 family, although downregulation of the anti-apoptotic Bcl-2 protein may have contributed to apoptosis at later time points. I3C caused a dose-dependent decrease in Akt phosphorylation and activity in the MDA MB468 cells, which possessed constitutive Akt activity caused by loss of PTEN expression. I3C was shown to similarly inhibit Akt phosphorylation cell line, but not in the high PTEN/low phospho-Akt expressing Du145 prostate cells or the HBL 100 line. I3C inhibited PI3K activity in the MDA MB468 cells (50mM) and HBL 100 cells (500mM) when added directly into an in vitro kinase assay. Induction of apoptosis in the MDA MB468 and LNCaP cells by the PI3K inhibitor LY294002 supported the hypothesis that inhibition of PI3K signalling may contribute to apoptosis. However, induction of apoptosis by I3C also occurred via an Akt-independent pathway that may involve NF-kB, as I3C, but not LY294002, decreased NF-kB DNA-binding (as shown by EMSA) in the MDA MB468 cells.

The naturally derived polyphenol curcumin has been investigated for prevention and treatment of many cancers over the past few decades. Accumulating evidence demonstrates that curcumin can target many tumorigenic pathways in cancer cells, rendering it a compound of interest for systematic investigation into prevention and treatment of an array of diseases, including non-small cell lung cancer (NSCLC). IC50 values for curcumin and first-line chemotherapeutic agents cisplatin and pemetrexed, were determined for A549, PC9 and PC9ER NSCLC cell lines. IC50s were then reassessed following long-term (>3 months), low-dose treatment with pharmacologically achievable curcumin doses to determine whether resistant subclones may develop. No significant changes in IC50 values were observed in any of the cell lines. Minor changes to expression of oncology-related proteins were concurrently observed, which reverted back to pre-treatment levels of expression following curcumin withdrawal. Cisplatin/pemetrexed double resistant cell lines were developed, and showed greater sensitivity to curcumin compared to their native cell line counterparts. Development of organotypic fibroblast co-cultures allowed assessment of cell invasion in a 3D model, creating better replication of cell-cell interactions within a more physiologically relevant environment. Co-culture models revealed that curcumin was able to inhibit invasion of NSCLC cells into a fibroblast-containing scaffold, with efficacy greatest at 0.25 - 0.5 μM curcumin, representing a non-linear dose response pattern. Furthermore, curcumin showed higher efficacy in supressing invasion of PC9 and PC9ER cisplatin/pemetrexed double resistant cell lines, reducing the area of invasion by up to 36% compared to that observed for their native non-resistant counterparts. In vivo, the bioavailable formulation of curcumin (Meriva) did not elicit significant effects on tumour multiplicity in the KRASG12D transgenic mouse model. However, decreased weight loss was observed in animals consuming Meriva, in addition to observing a decreased proliferative index in tumours compared to those animals fed control diet. Pharmacokinetic analysis demonstrated that dietary supplementation of 0.226% Meriva was sufficient to furnish lung tissue with detectable amounts of curcumin and its metabolites, proving that curcumin could be successfully delivered to the target tissues via an oral dosing regimen. This thesis has evaluated whether there may be potential for benefit or adverse effects of curcumin in lung cancer prevention regimens. Evidence presented suggests that long term curcumin treatment neither results in acquired resistance, nor causes resistance to standard-of-care chemotherapy agents. Furthermore, chemotherapy resistant subclones appear more sensitive to curcumin than their native counterparts, and in particular, curcumin is more potent in cells bearing EGFR mutational status compared to KRAS. Curcumin (delivered as the Meriva formulation) successfully reaches its target tissue (lung), and alleviates cancer associated weight loss in the KRAS mouse model.

Palliative treatment of metastatic colorectal cancer provides an overall median survival rate of approximately 21 months, with response rates of less than 60%. Curcumin is a low molecular weight polyphenol derived from the spice turmeric that inhibits carcinogenesis in vitro and in vivo via multi-targeted mechanisms. A clinical study was established investigating the safety and feasibility of administering curcumin with standard oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer and in parallel biomarker analysis was conducted to identify potential biomarkers of efficacy and toxicity. Methods: Phase I was a dose escalation phase using the traditional escalation rule (3+3+3) design to establish the maximum target dose of curcumin. Phase IIa was an open-labelled, two-armed, randomised controlled feasibility trial. Patients received standard oxaliplatin and 5-FU chemotherapy with or without the maximum target dose of curcumin established in Phase I. Biomarker studies were conducted involving measurement of miR-122, curcumin/curcuminoids and DNA platination in patient plasma samples, and proteomic analysis of treated explant media from patient-derived colorectal liver metastasis. Results: Phase I dose escalation was successfully completed with thirteen patients receiving curcumin plus standard oxaliplatin-based chemotherapy up to the target dose of 2 grams daily with no significant issues identified with toxicity or feasibility. Eighteen patients had been recruited into Phase IIa at the time of this report with no notable safety concerns. Changes in miRNA and curcumin/curcuminoid levels were successfully measured in patient plasma samples. Explant culture analysis showed proteins involved in apoptosis, angiogenesis and inflammation/immune response were selectively upregulated following treatment with CUFOX. Conclusion: Addition of curcumin to standard FOLFOX chemotherapy up to a dose of 2 grams daily has shown good tolerability, feasibility and no safety concerns across Phase I and IIa of this study. Potential biomarkers for future investigation have been identified.

Lung cancer is a leading cause of cancer deaths and is commonly diagnosed in both males and females. The cause of lung cancer is mainly attributable to cigarette smoking and aging, accounting for more than half of all lung cancer deaths. Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer (about 80%) compared to small cell lung cancer (SCLC) and it is normally subclassified into squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Cisplatin-based treatments are the main first-line regimens for any stage of NSCLC, although platinum has over a 40-year history of use in clinical practice. Recently, molecularly targeted agents have been developed for improvement of treatment outcomes in patients with advanced lung cancers, such as EGFR-and ALK-targeted drugs. However, the acquired resistance to these agents eventually impairs sensitivity to treatment. Studies have shown that PI3K/Akt signaling activation is associated with resistance to the targeted treatments, and highlights a role for targeting PI3K/Akt signaling to restore sensitivity to targeted therapies. Moreover, Akt inhibition has been shown to promote sensitivity to cisplatin treatment. In this project, both cisplatin and various agents which target Akt directly or indirectly, were tested on 2D NSCLC cell cultures for assessment of potencies. Single-agent treatment showed that the mutant P53 cell line (H596) showed the greatest sensitivity to cisplatin treatment compared to two WT P53 cell lines (A549 and H460). This was similarly observed in a patient-derived tissue explant model which showed that P53 mutant cases were more sensitive to cisplatin treatment than those WT P53 cases. In 2D adherent cell culture and 3D organotypic co-culture of cancer cells with fibroblasts, Akt activity was activated in response to cisplatin treatment, which was similarly observed in one explant case. Moreover, decreased Akt phosphorylation was significantly correlated with increased PARP cleavage in WT P53 cases only. Both PI3K- and Akt-targeted treatments were shown to be highly potent on 2D culture and 3D spheroid models compared to other cisplatin-based regimens. Further analysis of molecular biomarkers by In-Cell western assay corroborated this, with induction of caspase-3-dependent cell death significantly higher when cisplatin treatment was combined with PI3K- or Akt-targeting drugs. The increase in apoptosis was concurrently observed with decreases in pAkt levels. P53 and PTEN levels were not induced by combination treatments compared to cisplatin alone, suggesting that functional P53 might not be required for induction of cell death.

Curcumin, a naturally occurring dietary polyphenol, has been investigated for several years for its role in chemoprevention of cancer. There are emerging evidences that curcumin may have a potential role in prevention and treatment of lung cancer. Curcumin in its natural form is poorly absorbed, extensively metabolised and rapidly excreted. Meriva, a phospholipid formulation of curcumin, promises to enhance its bioavaibility. The work described in this thesis investigates whether Meriva offers pharmacokinetic advantage over unformulated curcumin, elucidate a possible mechanism of action for curcumin and determine efficacy of Meriva in vivo using xenograft model of lung cancer. Meriva offered superior curcumin and curcumin metabolites levels in mice plasma and lungs at two different dose levels (high and low). The levels were 5-16 fold higher for Meriva as compared to unformulated curcumin. A HPLC-UV method developed and validated for simultaneous quantification of curcumin and metabolites was found to reliable and reproducible. In vitro, a 3D organotypic model was used to demonstrate important role stromal components, namely fibroblasts, play in cell invasion and metastasis. Curcumin treatment of organotypic cultures significantly inhibited tumour cell invasion. Curcumin also modulated a number of key proteins in HGF/MET signalling axis. Nude mice were administered either control diet or diet supplemented with 0.226% w/w Meriva one week after A549:MRC5 (1:5) cell inoculation. Meriva significantly inhibited tumour growth versus control (p<0.05). The expression of p53, Ki-67 and cleaved caspase-3 which were significantly altered following Meriva intervention. These results together provide evidence that Meriva at a clinical relevant dose regime may be investigated in prevention and treatment of lung cancer.

Colorectal cancer remains the third largest cause of mortality from cancer related death, with approximately 90% of these deaths attributed to metastatic spread of the disease. There is a need to improve chemotherapy, with the dietary polyphenol curcumin, derived from turmeric, representing a potential candidate as it possess very few side effects and it has shown efficacy in mouse models. Recent advances in our understanding of tumour development have highlighted the existence of tumour initiating cells (TIC), which possess clonogenic potential, are essential for tumour growth, and represent an important therapeutic target. This study sought to determine whether curcumin in combination with oxaliplatin+5-Fluorouracil (OX+5-FU) represented a better combination for targeting TICs, using a variety of models consisting of cells derived directly from colorectal liver metastasis (CRLM). ALDHHigh activity, CD133 and CD26 were all found to mark a spheroid forming population, a method that tests for clonogenicity and selects for TICs. Curcumin significantly reduced the number of spheroids compared to DMSO, and enhanced efficacy of OX+5-FU. The only marker to decrease after treatment was ALDHHigh activity, which was also positively associated with spheroid growth. CD26 expressing cells were identified as a possible chemo-resistant population, however, this population remained unaffected by curcumin. Ex vivo analysis using explant cultures demonstrated that curcumin could significantly decrease ki67 and increase cleaved capase-3 expression, notably enhancing the effects of oxaliplatin in a proportion of patients. A pilot study was undertaken using non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice to reflect a clinical regimen, and assess in vivo whether curcumin could enhance efficacy of OX+5-FU, but the results advocate the use of higher doses as little effect was seen. Overall this body of work contributes to knowledge on propagating CRLM TICs, their expression of known TIC markers, and response to curcumin.

FOLFOX (5FU/leucovorin/oxaliplatin) is standard first-line therapy for metastatic colorectal cancer (mCRC), with response rates of 50%. Factors limiting the use of oxaliplatin include chemo-resistance and severe dose-related peripheral neuropathy. New interventional strategies are required which can improve disease outcomes by increasing drug efficacy whilst avoiding toxicity. Curcumin is a constituent of turmeric. Mechanisms of chemopreventive action, extensively investigated in vitro and in vivo, provide compelling evidence that curcumin exhibits efficacy in preventing neoplastic conditions and can act synergistically with chemotherapy. Laboratory endpoints must be tested clinically. In preparation for a clinical trial combining curcumin with FOLFOX, 3 lines of investigation have been followed. In vitro analysis of CRC cell lines treated with the investigative agents has explored mechanisms of chemo-resistance and the efficacy of curcumin therein. The acceptability of oral curcumin to patients has been assessed, supported by sensitive Ultra Performance Liquid Chromatography (UPLC) evaluation of curcumin and its metabolites in biomatrices. Parallel methods of biomarker profiling of plasma and urine from patients with CRC by NMR, LC-MS/MS and HD-MS have been tested for their suitability as adjuncts to trials studying mCRC. The resultant clinical method, guided by these components, devised for the assessment of curcumin and FOLFOX has been presented alongside early trial data. In vitro, curcumin was observed to overcome oxaliplatin-resistance and increase efficacy. Mechanisms of action have been proposed and sub-populations of stem-like cells suggested as potential treatment targets. Safety and tolerability of a suitable curcumin regimen have been shown clinically. Sensitive analysis of plasma, urine and tissue curcuminoids has been achieved, and prolonged mucosal exposure revealed. The proteomic and metabonomic profiles of patients with CRC are distinct from controls and could feasibly be employed as surrogate endpoints to trials. A dose-escalation study has endorsed an acceptable regimen for an on-going randomised trial investigating curcumin with FOLFOX (“CUFOX”).