2. HDAC6 : HDAC6 is a cytoplasmic enzyme that regulates many important biological processes. : HDAC6 has recently emerged as a tubulin deacetylase that has effects on microtubule- mediated processes. Zinc finger domain

3. HDAC6 deacetylates α-tubulin and associates with dynein. microtubules using the dynein motor complex.

4. 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. degradation

5. HDAC6 inhibitors - tubacin

6. topoisomerase Ⅱ inhibitor – toposide, doxorubicin During replication, DNA can become supercoiled. So, etopoisomerase Ⅱ preventing snarling. topoisomerase inhibitors block the ligation step of the cell cycle, generating single and double stranded breaks.

7. 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.

8. 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

9. HFS Tubacin reduced the rate of growth of transformed and, to a lesser extent, normal cells, without loss of cell viability

10. 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

11. In LNCaP cells, culture with the combination of 2.5 μM SAHA plus 8 μM tubacin resulted in an 80% loss of cell viability after 72 h. ☞

12. 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. LNCaP

13. 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. MCF-7 : human adenocarcinoma cells

14. To evaluate whether the effect of tubacin in enhancing the cytotoxic 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. LNCaP

15. Nil-tubacin did not increase cell death of LNCaP in combination culture with SAHA, etoposide, or doxorubicin.

16. They next determined whether LNCaP cells in whichHDAC6 expression was genetically suppressed were more sensitive than WT cells to SAHA-, etoposide-, or doxorubicin-induced cell death. ☞

17. Knockdown of HDAC6 resulted in a decrease in the rate of cell growth. Knockdown of HDAC6 did not affect cell viability.

18. HDAC6 knockdown of LNCaP cells cultured with SAHA for 48 h.

19. 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.

20. To investigate the pathway of cell death in LNCaP cells cultured with the combination of tubacin and SAHA or toposide. 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

21. Cells cultured with 5 μM SAHA resulted in PARP cleavage. cells cultured with the combination of tubacin and etoposide induced PARP degradation.

22. To further examine caspase-dependent activation in cells cultured with tubacin and SAHA or etoposide. the pan-caspase inhibitor Z-VAD-fmk was added.

23. 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.

24. They examined selective inhibition of HDAC6 with tubacin activated a DNA damage response. ☞ confirmed that in LNCaP cells increased accumulation of γH2AX.

25. Quantitation of γH2AX levels. tub+SAHA 6-fold incresed. tub+etoposide 1.5-fold incresed.

26. They confirmed the activation of the checkpoint kinase Chk2. Culture with SAHA or etoposide alone resulted in the activation of Chk2, as shown by an increase of phospho-Chk2.

27. They confirmed the activation of the checkpoint kinase Chk2. ☞ HDAC6 inhibition enable to the DNA damage and checkpoint response induced by SAHA or etoposide.

28. 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. Table S2. ☞☞

29. Culture with SAHA alone did not induce DDIT3 at 8 h or 24 h. Table S2. ☞

30. Increased expression of DDIT3 was confirmed on analysis at the protein level. ☞ So, Tubacin up-regulates DDIT3 and DDIT4, down-regulates replication proteins.

31. Expression of several genes regulating replication progression in cultured with tubacin, SAHA, or etoposide alone and in combination. Mcm4, Mcm6, Cdt1, and Psf2 : DNA replication factors

32. Cells cultured for 24 h as described in → These inhibitors suppress DNA replications.

33. 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 Caspase- 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.