ATP-binding cassette subfamily C (ABCC) transporter 1 (ABCC1) and 4 (ABCC4) independent of their drug efflux ability affects breast cancer biology

Breast cancer treatment has been a challenge to date, due in part to cancer cells acquiring drug resistance. One of the mechanisms by which resistance can occur is the overexpression of drug efflux pumps such as ATP-binding cassette, subfamily C (ABCC) transporter 1(ABCC1) and 4 (ABCC4), which are members of ABC transporters. Recently research has shown that these proteins may be implicated in cancer biology independent of cytotoxic drug efflux, but so far little is known about this in regards to breast cancer. ABCC1 and ABCC4 protein levels in MDA-MB231 and MCF-7, human breast cancer derived cell lines were measured by Western blot. The role of ABCC1 and ABCC4 in cell proliferation and migration were evaluated by colony formation, MTT and scratch assays in the presence of various ABCC inhibitors – MK571, Indomethacin, Reversan, Ceefourin 1 and Ceefourin 2, inhibitors of ABCC4. Similarly the effect on proliferation, migration and invasion was monitored following knock down of ABCC1 and ABCC4 with ABCC1- and ABCC4- specific siRNAs, or overexpression using transfection with pcDNA3.1 plasmids containing the ABCC1 and ABCC4 genes. The potential correlation between ABCC1 and/or ABCC4 and the expression of Gprotein-coupled receptor 55 (GPR55), or extracellular signal-regulated kinase (ERK), or the extracellular efflux of cyclic adenosine monophosphate (cAMP), prostaglandin E2 (PGE2), sphingosine-1-phosphate (S1P) and cyteinyl leukotrienes (LTC4, LTD4 and LTE4) were investigated by Western blot and enzyme-linked immunosorbent assays. This thesis demonstrates that the expression levels of ABC transporters varies between breast cancer cell lines. Our results suggest that ABCC1 may be more involved in mediating breast cancer cell proliferation than ABCC4 and in contrary ABCC4 may be involved in mediating breast cancer cell invasion more than ABCC1. There is also an indication that both ABCC1and ABCC4 are implicated in breast cancer migration. In addition, potential correlation between ABCC1 and/or ABCC4 with cAMP or S1P efflux looks promising but further investigation is required. Taken together this thesis shows that ABCC1 and ABCC4 may be implicated in breast cancer development and progression. Further investigations are needed to validate our current results, but ABCC1 and ABCC4 could be potential therapeutic targets for breast cancer

Roles of ABCC1 and ABCC4 in Proliferation and Migration of Breast Cancer Cell Lines

ABCC1 and ABCC4 utilize energy from ATP hydrolysis to transport many different molecules, including drugs, out of the cell and, as such, have been implicated in causing drug resistance. However recently, because of their ability to transport signaling molecules and inflammatory mediators, it has been proposed that ABCC1 and ABCC4 may play a role in the hallmarks of cancer development and progression, independent of their drug efflux capabilities. Breast cancer is the most common cancer affecting women. In this study, the aim was to investigate whether ABCC1 or ABCC4 play a role in the proliferation or migration of breast cancer cell lines MCF-7 (luminal-type, receptor-positive) and MDA-MB-231 (basal-type, triple-negative). The effects of small molecule inhibitors or siRNA-mediated knockdown of ABCC1 or ABCCC4 were measured. Colony formation assays were used to assess the clonogenic capacity, MTT assays to measure the proliferation, and scratch assays and Transwell assays to monitor the cellular migration. The results showed a role for ABCC1 in cellular proliferation, whilst ABCC4 appeared to be more important for cellular migration. ELISA studies implicated cAMP and/or sphingosine-1-phosphate efflux in the mechanism by which these transporters mediate their effects. However, this needs to be investigated further, as it is key to understand the mechanisms before they can be considered as targets for treatment

The synthesis of recombinant membrane proteins in yeast for structural studies

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies

Phase II trial of sagopilone, a novel epothilone analog in metastatic melanoma

BackgroundSagopilone is a novel fully synthetic epothilone with promising preclinical activity and a favourable toxicity profile in phase I testing.MethodsA phase II pharmacokinetic and efficacy trial was conducted in patients with metastatic melanoma. Patients had measurable disease, Eastern Cooperative Oncology Group performance status 0-2, adequate haematological, and organ function, with up to 2 previous chemotherapy and any previous immunotherapy regimens. Sagopilone, 16 mg m⁻², was administered intravenously over 3 h every 21 days until progression or unacceptable toxicity.ResultsThirty-five patients were treated. Sagopilone showed multi-exponential kinetics with a mean terminal half-life of 64 h and a volume of distribution of 4361 l m⁻² indicating extensive tissue/tubulin binding. Only grade 2 or lower toxicity was observed: these included sensory neuropathy (66%), leukopenia (46%), fatigue (34%), and neutropenia (31%). The objective response rate was 11.4% (one confirmed complete response, two confirmed partial responses, and one unconfirmed partial response). Stable disease for at least 12 weeks was seen in an additional eight patients (clinical benefit rate 36.4%).ConclusionSagopilone was well tolerated with mild haematological toxicity and sensory neuropathy. Unlike other epothilones, it shows activity against melanoma even in pretreated patients. Further clinical testing is warranted