Proteomics(Cancer cell) Proteomic investigation of taxol and taxotere resistance and invasiveness in a squamous lung carcinoma cell line L. Murphy 외 Proteomics(Cancer cell) 생명과학기법 과제 – 3 20641022 이용수

Introduction 5% survival for 5 years Lung cancer

Introduction death Early diagnosis Surgery - survival Late stage diagnosis lethality↑ death

anti-neoplastic effects Introduction Taxanes anti-neoplastic effects ovarian breast melanomas small-cell and non-small-cell lung cancers

Introduction Micro tubule resistance increased expression of the efflux pump P-gp alterations in the expression of tubulins mutations at the taxane site of action in microtubules expression of apoptosis-related proteins

maintenance of cell shape Introduction microtubule mitosis spindle poisons maintenance of cell shape

two-dimensional electrophoresis Introduction two-dimensional electrophoresis spectrometry mass SKMES-1 altered protein expression in the human squamous lung carcinoma cell line

Materials & Methods Toxicity testing Cells were set up in growth medium Cytotoxic drug dilutions The plates were incubated The plate was read at 405 nm

Two Dimensional-Difference in Gel Electrophoresis Materials & Methods Two Dimensional-Difference in Gel Electrophoresis Lysates of exponentially growing cells were minimally labeled with Cy3 or Cy5 Reverse labeling was carried out Proteins were defined as differentially regulated

Protein identification Materials & Methods Protein identification by mass spectrometry Differentially expressed proteins were identified picked using an Ettan Spot Picker In-gel digestion, vacuum-dried Peptide mass fingerprinting for protein identificationwas performed

Materials & Methods Western blotting Western blotting was performed on cell lysates prepared for DIGE Samples were separated on a 15% SDS gel Secondary antibodies conjugated to horse-radish peroxidase were detected Western blots were analysed

Materials & Methods Invasion assays modified version of the Boyden chamber invasion assay The level of invasion was determined microscopically counting the number of cells Adhesion assays Plate was read at 405 nm

Toxicity profile of SKMES-1 and taxane-selected variants Results Toxicity profile of SKMES-1 and taxane-selected variants

Role of P-gp in resistance Results Role of P-gp in resistance P-gp expression in SKMES-1 and taxane-selected variants at weeks 2, 5 and 6 in culture

DIGE analysis of SKMES-1 versus SKMES-1-Taxotere Results DIGE analysis of SKMES-1 versus SKMES-1-Taxotere Differentially expressed proteins between SKMES-1 and Taxane-selected variants correlating with increased resistance and invasion. showing differentially expressed proteins

Differential protein expression of taxol-resistant variants of SKMES-1 Results Differential protein expression of taxol-resistant variants of SKMES-1

DIGE analysis of SKMES-1 versus SKMES-1-Taxol Results DIGE analysis of SKMES-1 versus SKMES-1-Taxol Ontology analysis of identified differentially regulated proteins Identified to date according to function Tumour immunity, apoptosis, mitochondrial processing and cytokinesis

Invasion and adhesion assays Results Invasion and adhesion assays Quantification of invasion of SKMES-1 and taxane-resistant variants through matrigel

Results Quantification of adhesion of SKMES-1 and taxane-resistant variants to matrigel

Modulation of stress response proteins Discussion Modulation of stress response proteins Proteomic analysis VCP signal target of Akt required for cell survival oxygen-regulated protein Taxotere HSP70A8 and HSP60, CCT3 and HSPC124 were increased taxone HSP70A8, HSP27 and HSP70 9B were all down

Discussion up-regulated in both increased in SKMES-1-Taxotere Prohibitin (PHB) up-regulated in both Cathepsin D increased in SKMES-1-Taxotere found to be increased in many cancer not in lung cancer

Reactive oxygen species (ROS) Discussion Reactive oxygen species (ROS) ROS leading to irreversible cell injury Both taxane-resistant variants showed protective effects against ROS ER-60 cellular detoxification proteins act as a protective response against DNA damage

Modulation of invasion-related proteins Discussion Modulation of invasion-related proteins increase Expression of cytoskeletal proteins Taxol selection decrease

Modulation of invasion-related proteins Discussion Modulation of invasion-related proteins SKMES-1-Taxotere & SKMES-1-Taxol6 Increase in high mobility group box protein nuclear complex formation apoptosis drug resistance

Modulation of invasion-related proteins Discussion Modulation of invasion-related proteins increase expression of Ubiquitin Carboxy Hydrolase L1 Taxane selection associated with advanced stage lung cancers as marker for aggressive and invasive colorectal carcinoma

Further Thinking 특정한 약에 대해 반응하는 기전에 의해 변화하는 특정 유전자의 발현 정도를 가지고 stage를 알아보고자 하는 방법이 매우 획기적이었다. 항암제에 대한 resistance도 그 이유에 대해 연구하면 많은 결과를 얻을 수 있을 것으로 보인다. 하지만 이러한 우회적인 방법은 획기적이기는 하지만 다른 변수가 포함될 수 있어 신뢰도를 확인할 수 있는 방법을 좀더 체계적으로 고안해야 한다. Cancer 의 성향이 각 부위마다 다르다는 것을 바탕으로 연구를 했기 때문에 이에 대한 사전의 연구가 기본이 되어 있다는 것을 예측할 수 있다. 이러한 기본연구가 역시 중요한 듯 보인다.