1. 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
이용수

2. Introduction
5% survival for 5 years
Lung cancer

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

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

5. 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

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

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

8. 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

9. 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

10. 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

11. 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

12. 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

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

14. 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

15. 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

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

17. 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

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

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

20. 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

21. 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

22. 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

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

24. 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

25. 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

26. 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.