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Introduction Methodology
Rat breast adenocarcinoma cells, MTLn3 cells were grown on high-density (20 mg/ml) 3D collagen matrix in complete AdvDMEM without
In cancer, reduced histone acetylation is significantly correlated
antibiotics. Twenty four hour after seeding, cells were treated with different concentration of MS-275 alone or combination with other drugs for
with advanced tumor stages and depth of the cancer invasion
(1). Histone deacetylases (HDACs) have been recently shown to 48hr.
be important factors in cell migration and invasion for both normal Confocal microscopy: forty-eight hours after MS-275 treatment, cells were fixed in 4% PFA and stained in Alexa fluor 488 phalloidin. Cells were
and malignant cells. HDAC inhibitors are being used in clinical Imaged under the Olympus FV1000 confocal microscope. Live cell images were taken in GFP-actin transfected cells.
trails to counter cancer and they function by blocking cell Extraction of total RNA and Q-PCR: Total RNA was extracted using Trizol and RNeasy Mini extraction kit. One micro gram of total RNA was
proliferation, angiogenesis and cell invasion and by promoting reverse transcribed using Superscript111 First strand synthesis system and 1µl of cDNA was used to quantify genes in Rotor gene 6000.
cell differentiation and apoptosis (2). In our study, the class I Protein extraction and ROCK activity assay : HD gels were digested in 0.5mg/ml Collagenase, supplemented with 1X Kerb’s ringer buffer and
HDAC inhibitor, MS-275, significantly downregulates the 100mM CaCl2 for 30min at 37oC. Cells were pelleted at 2000rpm and washed 2 times in cold PBS. Cells were lysed in RIPA buffer and
metastasis promoter gene ROCK1 in mRNA, protein level and
supernatant was separated at 13,000rpm for 15min. Ten microgram of protein was used to measure activity using ROCK activity assay kit from
activity levels. This effect is mediated by intermediate molecules
(p53 and Notch). Cell Biolabs Inc. In Brief, 10µg of protein was incubated in 65ul of 1X Kinase/ ATP/ ROCK substrate solution (MYPT1) at 37oC for 30min.
Protein/substrate was separated 12% SDS gels.
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MS-275 increased the number and size of MS-275 increased the expression of RhoB and N-WASP,
Live cell imaging showing protrusion- and
protrusions in MTLn3 cells. genes that function in cell migration.
contractility-led invasion mechanisms.
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Relative ROCK1 α -tubulin
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0.8 0.6 MS-275
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0.6 0.4
expression 0.2 for the inhibition of
0.4 Relative
0.2 p53 expression
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Relative ROCK1
ROCK1; drug-ablation
0
Control 1uMMS- 3uMMS- of p53 or Notch1
Control 1uM MS- 3uM MS-
275 275 275 275 Control reversed the effects of
3uMMS-275 10uMDAPT MS-275 on ROCK1
10uMPifithrin MS-275&DAPT
MS-275 increased the transcripts of the tumour suppressors, Notch1 and p53, in MS-275&Pifithrin levels.
concentration dependant manner.