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• HDAC6
: HDAC6 is a cytoplasmic enzyme that regulates many im-portant biological processes.
: HDAC6 has recently emerged as a tubulin deacetylase that has effects on microtubule- mediated processes.
INTRODUCTION
Zinc finger do-main
INTRODUCTION
• HDAC6 deacetylates α-tubu-lin and associates with dynein.
• microtubules using the dynein motor complex.
• HDAC inhibitor(HDACi) : accumulation of acetylated forms of proteins which can
alter their structure and function.
. Normal cells are resistant to HDACi induced cell death. But HDACi can induce different phenotypes in transformed
cells, including growth arrest, apoptosis.
INTRODUCTION
degradation
INTRODUCTION• HDAC6 inhibitors - tubacin
INTRODUCTION• topoisomeraseⅡ inhibitor – toposide, doxorubicin• During replication, • DNA can become supercoiled.• So, etopoisomeraseⅡ • preventing snarling.
• • topoisomerase inhibitors • block the ligation step of • the cell cycle, generating • single and double stranded • breaks.
• pan-HDAC inhibitor - SAHA : it is grouped into HDAC inhibitor categories, pan-HDAC inhibitor decrease all class I and II HDAC isoforms. representatively, there are TSA and SAHA.
INTRODUCTION
RESULT-TUBACIN ENHANCES TRANSFORMED BUT NOT
NORMAL CELL DEATH INDUCED BY TOPOISOMERASE II IN-HIBITORS AND A PAN-HDAC INHIBITOR.
• acetylated α-tubulin and acetylated histone H3 in HFS cells and LNCap cells cultured for 24 h with tubacin, SAHA, and tubacin + SAHA.
Fig 1. Fig 2.
HFS cells LNCaP cells
RESULT-TUBACIN ENHANCES TRANSFORMED BUT NOT
NORMAL CELL DEATH INDUCED BY TOPOISOMERASE II IN-HIBITORS AND A PAN-HDAC INHIBITOR.HFS HFS
• Tubacin reduced the rate of growth of transformed and, to a lesser extent, normal cells, without loss of cell viability
• To assess whether specific inhibition of HDAC6 enhances cell death when combined with anticancer agents in HFS cells.
→ tubacin had no detectable effect on cell viability
RESULT-TUBACIN ENHANCES TRANSFORMED BUT NOT
NORMAL CELL DEATH INDUCED BY TOPOISOMERASE II IN-HIBITORS AND A PAN-HDAC INHIBITOR.
• In LNCaP cells, culture with the combination of 2.5 μM SAHA plus 8 μM tubacin resulted in an 80% loss of cell viability af-ter 72 h.
RESULT-TUBACIN ENHANCES TRANSFORMED BUT NOT
NORMAL CELL DEATH INDUCED BY TOPOISOMERASE II IN-HIBITORS AND A PAN-HDAC INHIBITOR.
☞
• LNCaP cell death was markedly enhanced in cultures with tubacin and 25 μM or 50 μM etoposide. Similarly,
• LNCaP cell death was enhanced in culture with tubacin plus doxorubicin.
RESULT-TUBACIN ENHANCES TRANSFORMED BUT NOT
NORMAL CELL DEATH INDUCED BY TOPOISOMERASE II IN-HIBITORS AND A PAN-HDAC INHIBITOR.
LNCaP
• Tubacin increased the sensitivity of MCF-7 cells to SAHA-, etoposide-, and doxorubicin-induced cell death.
☞ these data shows that tubacin and combination with SAHA, etoposide, doxorubicin gives an effect to only transformed cell.
RESULT-TUBACIN ENHANCES TRANSFORMED BUT NOT
NORMAL CELL DEATH INDUCED BY TOPOISOMERASE II IN-HIBITORS AND A PAN-HDAC INHIBITOR.
• MCF-7 : human adenocarcinoma cells
• To evaluate whether the effect of tubacin in enhancing the cyto-toxic effects of SAHA, etoposide, or doxorubicin.
• LNCaP cells were cultured with nil-tubacin
☞ nil-tubacin ?
: an analogue of tubacin that does not inhibit HDAC6 deacetylase activity.
RESULT-TUBACIN ENHANCES TRANSFORMED BUT NOT
NORMAL CELL DEATH INDUCED BY TOPOISOMERASE II IN-HIBITORS AND A PAN-HDAC INHIBITOR.
LNCaP
• Nil-tubacin did not increase cell death of LNCaP in combination culture with SAHA, etoposide, or doxorubicin.
RESULT-TUBACIN ENHANCES TRANSFORMED BUT NOT
NORMAL CELL DEATH INDUCED BY TOPOISOMERASE II IN-HIBITORS AND A PAN-HDAC INHIBITOR.
• They next determined whether LNCaP cells in whichHDAC6 ex-pression was genetically suppressed were more sensitive than WT cells to SAHA-, etoposide-, or doxorubicin-induced cell death.
RESULT-DOWN-REGULATION OF HDAC6 EXPRESSION IN
LNCAP INCREASES SENSITIVITY TO CELL DEATH INDUCED BY SAHA, ETOPOSIDE, OR DOXORUBICIN.
☞
• Knockdown of HDAC6 resulted in a decrease in the rate of cell growth.
• Knockdown of HDAC6 did not affect cell viability.
RESULT-DOWN-REGULATION OF HDAC6 EXPRESSION IN
LNCAP INCREASES SENSITIVITY TO CELL DEATH INDUCED BY SAHA, ETOPOSIDE, OR DOXORUBICIN.
• HDAC6 knockdown of LNCaP cells cultured with SAHA for 48 h.
RESULT-DOWN-REGULATION OF HDAC6 EXPRESSION IN
LNCAP INCREASES SENSITIVITY TO CELL DEATH INDUCED BY SAHA, ETOPOSIDE, OR DOXORUBICIN.
• Increased sensitivity to etoposide- or doxorubicin-induced cell death was observed in LNCaP cells with reduced HDAC6 expression.
☞ chemical inhibition of HDAC6 or genetically reduced HDAC6 expression increases the sensitivity of LNCaP cells
to SAHA-, etoposide-, or doxorubicin- induced cell death.
RESULT-DOWN-REGULATION OF HDAC6 EXPRESSION IN
LNCAP INCREASES SENSITIVITY TO CELL DEATH INDUCED BY SAHA, ETOPOSIDE, OR DOXORUBICIN.
• To investigate the pathway of cell death in LNCaP cells cul-tured with the combination of tubacin and SAHA or topo-side. So They performed PARP assay.
• PARP assay : PARP is a 116-kDa nuclear protein that is specifically cleaved by caspase- 3 into a 85-kDa fragment and serves as a marker of apoptosis
RESULT-ACTIVATION OF APOPTOTIC PATHWAY IS EN-
HANCED IN TRANSFORMED CELLS.
• Cells cultured with 5 μM SAHA resulted in PARP cleavage.
• cells cultured with the combination of tubacin and etoposide induced PARP degradation.
RESULT-ACTIVATION OF APOPTOTIC PATHWAY IS EN-
HANCED IN TRANSFORMED CELLS.
• To further examine caspase-dependent activation in cells cultured with tubacin and SAHA or etoposide.
• the pan-caspase inhibitor Z-VAD-fmk was added.
RESULT-ACTIVATION OF APOPTOTIC PATHWAY IS EN-
HANCED IN TRANSFORMED CELLS.
• LNCaP cells cultured with tubacin in combination with etoposide from 65% to 25%.
☞ cell death induced by the combination of tubacin and SAHA or
tubacin and etoposide is, in part, dependent on caspase
activation.
RESULT-ACTIVATION OF APOPTOTIC PATHWAY IS EN-
HANCED IN TRANSFORMED CELLS.
• They examined selective inhibition of HDAC6 with tubacin activated a DNA damage response.
☞ confirmed that in LNCaP cells increased accumulation of
γH2AX.
RESULT-TUBACIN ENHANCES THE ACCUMULATION OF
ΓH2AX AND PHOSPHO-CHK2 INDUCED BY SAHA OR ETOPOSIDE.
• Quantitation of γH2AX levels.
tub+SAHA 6-fold incresed.
tub+etoposide 1.5-fold incresed.
RESULT-TUBACIN ENHANCES THE ACCUMULATION OF
ΓH2AX AND PHOSPHO-CHK2 INDUCED BY SAHA OR ETOPOSIDE.
• They confirmed the activation of the checkpoint kinase Chk2.
• Culture with SAHA or etoposide alone resulted in the activa-tion of Chk2, as shown by an increase of phospho-Chk2 .
RESULT-TUBACIN ENHANCES THE ACCUMULATION OF
ΓH2AX AND PHOSPHO-CHK2 INDUCED BY SAHA OR ETOPOSIDE.
• They confirmed the activation of the checkpoint kinase Chk2.
☞ HDAC6 inhibition enable to the DNA damage and check-point
response induced by SAHA or etoposide.
RESULT-TUBACIN ENHANCES THE ACCUMULATION OF
ΓH2AX AND PHOSPHO-CHK2 INDUCED BY SAHA OR ETOPOSIDE.
• To characterize the molecular pathways altered by tubacin, SAHA, and the combination(tubacin + SAHA) on LNCaP cells.
They cultured with SAHA, tubacin, combination alone.
☞ DDIT4(DNA-damage-inducible transcript 4) was induced to a similar level in LNCaP cells cultured for 24 h with tubacin plus SAHA.
RESULT- TUBACIN UP-REGULATES DDIT3 AND DDIT4,
DOWN- REGULATES REPLICATION PROTEINS, AND INDUCES A G1 ARREST.
Table S2.
☞ ☞
Culture with SAHA alone did not induce
DDIT3 at 8 h or 24 h.
Table S2.
☞
RESULT- TUBACIN UP-REGULATES DDIT3 AND DDIT4,
DOWN- REGULATES REPLICATION PROTEINS, AND INDUCES A G1 ARREST.
• Increased expression of DDIT3 was confirmed on analysis at the protein level.
☞ So, Tubacin up-regulates DDIT3 and DDIT4, down-regu-
lates replication proteins.
RESULT- TUBACIN UP-REGULATES DDIT3 AND DDIT4,
DOWN- REGULATES REPLICATION PROTEINS, AND INDUCES A G1 ARREST.
• Expression of several genes regulating replication progres-sion in cultured with tubacin, SAHA, or etoposide alone and in combination.
Mcm4, Mcm6, Cdt1, and Psf2 : DNA replication factors
RESULT- TUBACIN UP-REGULATES DDIT3 AND DDIT4,
DOWN- REGULATES REPLICATION PROTEINS, AND INDUCES A G1 ARREST.
• Cells cultured for 24 h as described in
→ These inhibitors suppress DNA replications.
RESULT- TUBACIN UP-REGULATES DDIT3 AND DDIT4,
DOWN- REGULATES REPLICATION PROTEINS, AND INDUCES A G1 ARREST.
• Tubacin Enhances the Accumulation of γH2AX and Phospho-Chk2
Induced by SAHA or Etoposide in transformed cells. (LNCaP).
• Culture with Tubacin Plus SAHA or Etoposide Enhances Cas-pase-
Dependent Apoptosis in LNCaP Cells.
• Tubacin Induces a G1 Arrest, Up-Regulates DDIT3 and DDIT4, and
Down-Regulates DNA Replication Proteins.
• combination therapy with a selective HDAC6 inhibitor and certain anticancer agents may be a strategy for therapy of sensitive tumors.
CONCLUSION