• 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