HDAC1 ( IC50 = 36 nM ); HDAC2 ( IC50 = 47 nM ); HDAC4 ( IC50 = 510 nM ); HDAC6 ( IC50 = 14000 nM )

Romidepsin treatment potently inhibits the neovascularization of chick embryos and adult mice in the Matrigel plug assay. AThe median survival times of mice with U-937 lymphoma are 30.5 (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively, after receiving romidepsin at 0.1–1 mg/kg twice a week (compared to 20 days in control mice).

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FK228 showed potent antiangiogenic activities in vivo [4]
The antiangiogenic activity of FK228 was also investigated in vivo, using a CAM assay. As shown in Figure 3, the FK228 produced a significant decrease in the developmental of angiogenesis in a chick embryo without any sign of thrombosis and hemorrhage. The inhibition of the angiogenesis of RA used as the positive control was 70.83 ± 6.22% (n = 32) and that of empty coverslips was 4.43 ± 4.43% (n = 45). However, FK228 completely inhibited the neovascularization of chick embryo (100 ± 0.00%, n = 16) without affecting any preexisting vessels. The antiangiogenic activity of FK228 was also confirmed by performing an established in vivo angiogenesis model, the Matrigel plug assay in an adult mouse.29 The Matrigel was injected subcutaneously into C57/BL6J mice, solidified in vivo and removed from the mice at 5 days after implantation. For histologic examination, image analysis was performed. As shown in Figure 4, control plugs in which the Matrigel was injected with the heparin alone had little vessel, but bFGF (100 ng/ml) enriched abundant vessels inside the plugs. However, FK228 strongly inhibited the bFGF-induced angiogenesis. Hb contents of the Matrigel plugs were also measured to quantify the functional vasculature. Control plugs showed 0.55 ± 0.44 g/dl of Hb (n = 5) and bFGF-containing Matrigel showed 12.94 ± 2.82 g/dl (n = 6). However, FK228 markedly reduced the Hb levels to 1.36 ± 0.25 g/dl (n = 5). These results demonstrated that FK228 potently inhibits angiogenesis in vivo and in vitro.

In the enzyme assay, 90 μL of the HDAC enzyme fraction extracted from 293T cells overexpressing HDAC1 or HDAC2 in the presence of increasing concentrations of Romidepsin is mixed with 10 μL of [³H]acetyl-labeled histones (25,000 cpm/10 μg). The mixture is then incubated at 37 °C for 15 minutes. For a minimum of 60 minutes, the enzyme reaction is linear. The addition of 10 μL of concentrated HCl stops the reaction. For the purpose of determining radioactivity, 0.9 mL of the solvent layer is added to 5 mL of aqueous counting scintillant II solution after the released [³H]acetic acid is extracted with 1 mL of ethylacetate. At least three separate independent dose-response curves are used to calculate the IC50 values.

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[3H]-Methylthymidine assay [4]
To measure cell proliferation, the BAECs were placed in a 24-well culture plates at a density of 3 × 104 cells. Then, Romidepsin (FK228) was added and incubated under normoxic or hypoxic condition for 24 hr. Then 1 μCi [3H]-methylthymidine (25 mCi/mmol) was added at the final 4 hr before the assay. The cells were washed with PBS and fixed with methanol on ice for 30 min. Unincorporated [3H]-methylthymidine was removed by washing with 10% trichloroacetic acid (TCA). After the overnight incubation with 10% TCA, the cells were solubilized in 0.2 M NaOH and 0.1% SDS at 37°C for 1 hr. The counts per minute were determined with a liquid scintillation counter and the values were expressed as mean cpm (of triplicate). Each sample was assayed in triplicate and the experiment was repeated twice independently.

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HDAC activity assay [4]
The BAECs (1 × 107 to 5 × 107) treated with Romidepsin (FK228) (1–10 ng/ml) for 24 hr were harvested and lysed in 1 ml HDAC buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100). [3H]-Acetylated histones were incubated with 100 μl of prepared crude cell extract for 2 hr at 37°C. The reaction was quenched with 1 M HCl and 0.16 M acetic acid. Released [3H]acetate was extracted with 600 μl of ethyl acetate and quantified by a scintillation counter.

In 96-well plates, cells are exposed to different doses of romidepsin for a duration of 72 hours. For four hours, 20 μL of a 5 mg/mL MTT solution in PBS is added to each well. To dissolve the formazan crystals, 170 μL of DMSO is added to each well after the medium has been removed. At 540 nm, the absorbance is calculated. Moreover, trypan blue is added to the cells, and the quantity of transparent (living) and blue (dead) cells is counted in a hemocytometer. Cells are incubated in a propidium iodide staining solution containing 0.05 mg/mL propidium iodide, 1 mM EDTA, 0.1% Triton X-100, and 1 mg/mL RNase A in PBS for 30 minutes in order to perform a cell cycle analysis. The suspension is then examined using a Becton Dickinson FACScan after being run through a nylon mesh filter.

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Cell growth assay [4]
The endothelial cell growth was determined by 3-(4,5-diphenylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The BAECs were grown at a density of 1.5 × 10~4 cells in a 24-well culture plates and incubated for 24 hr for stabilization. The medium was replaced with 1.5 ml fresh medium and Romidepsin (FK228) was added. After 24 and 72 hr, the medium was aspirated and the MTT assay was performed. The amount of MTT-formazan was determined as absorbance at 540 nm. Each sample was assayed in triplicate and the experiment was repeated twice independently.

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Tube formation assay [4]
Matrigel (250 μl in a concentration of 10 mg/ml) was placed in a 24-well culture plate and polymerized for 30 min at 37°C. The BAECs (5 × 10~5 cells) were seeded on the surface of the Matrigel. Then, the Romidepsin (FK228) (10 ng/ml) was added and incubated for 24 hr under normoxic or hypoxic condition. The morphologic changes in the cells were observed under a microscope and photographed at a ×40 magnification using ImagePro Plus software. Each sample was assayed in duplicate and the experiment was repeated twice independently.

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Chemomigration assay [4]
Chemomigration of the BAECs was performed using a 24-well transwell culture chamber with 8.0-μm-pore polycarbonate filter inserts. The lower side of the filter was coated with 10 μl type IV collagen (3 mg/ml) and air-dried for 1 hr. The BAECs (3 × 104 cells) were placed in the upper part of the filters and Romidepsin (FK228) (10 ng/ml) was applied to both sides of the filter. The cells were incubated at 37°C for 8 hr under normoxic or hypoxic condition and then fixed with methanol and stained with hematoxylin and eosin. The cells on the upper surface were carefully removed by rubbing with a cotton swab and mounted on slide glasses. The cell migration was determined by counting the whole cell numbers on a single filter using optical microscopy at a ×40 magnification. Each sample was assayed in duplicate and the experiment was repeated twice independently.

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Chemoinvasion assay [4]
The invasiveness of the BAECs was performed in vitro using a transwell chamber system with 8.0-μm-pore polycarbonate filter inserts. The lower side of the filter was coated with 10 μl of type IV collagen (3 mg/ml), whereas the upper side was coated with 10 μμl Matrigel (0.5 mg/ml). The BAECs (3 × 10~4 cells) were placed in the upper part of the filters and Romidepsin (FK228) (10 ng/ml) was treated in both parts. The cells were incubated at 37°C for 18 hr under normoxic or hypoxic condition and then fixed with methanol and stained with hematoxylin and eosin. The cell invasion was determined by counting the whole cell numbers in a single filter using optical microscopy at a ×40 magnification. Each sample was assayed in duplicate and the experiment was repeated twice independently.

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Cell adhesion assay [4]
A cell adhesion assay was performed as described below. Each well of a 24-well culture plate was coated with type IV collagen (5 μg/ml), vitronectin (1 μg/ml), fibronectin (1 μg/ml) or type I collagen (5 μg/ml) and incubated for 2 hr at 37°C. After washing with PBS, 3.0% BSA was added to each well for 1 hr to prevent nonspecific attachment. The BAECs (3 × 10~4 cells) suspended in serum-free media was added to each coated well and Romidepsin (FK228) (10 ng/ml) was added. After incubation at 37°C for 1 hr under normoxic or hypoxic condition, non-adherent cells were washed off by streaming PBS over the plate 3 times. The remaining adherent cells were stained with 1% crystal violet and washed with PBS several times to remove the excess dye. The stained crystal violet was eluted with 10% acetic acid and determined by scanning with an ELISA reader with a 600-nm filter. Each sample was assayed in triplicate and the experiment was repeated twice independently.

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Chorioallantoic membrane (CAM) assay [4]
Fertilized chick eggs were kept in a humidified incubator at 37°C for 3 days. About 2 ml of egg albumin was then removed with a hypodermic needle allowing the CAM and yolk sac to drop away from the shell membrane. On day 3.5, the shell was punched out and removed and the shell membrane peeled away. At the stage of a 4.5-day old chick embryo, an Romidepsin (FK228)-loaded (50 ng/egg) Thermanox coverslip was air-dried and applied to the CAM surface. Two days later, 2 ml of 10% fat emulsion was injected into the chorioallantois and the CAM was observed under a microscope. Because retinoic acid (RA) is known as an antiangiogenic compound, 1 μg/egg RA was used as a positive control for antiangiogenic responses. When the CAM treated with Romidepsin (FK228) had an avascular zone to a similar degree as RA-treated CAM, the response was scored as positive and calculated based on the percentage of positive eggs to the total number of eggs tested. The experiment was repeated 3 times and more than 20 eggs were used each time.

Male scid mice inoculated i.p. with U-937 cells
~1 mg/kg once or twice a week
Treated i.p.

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In vivo mouse Matrigel plug assay [4]
The mouse Matrigel plug assay was performed as previously described. Briefly; C57BL/6J mice were injected subcutaneously with 0.5 ml Matrigel containing the indicated amount of Romidepsin (FK228) (0.7 μg/ml) with bFGF (100 ng/ml) and heparin (10 U/ml). The injected Matrigel rapidly formed a single, solid gel plug. After 5 days, mice were sacrificed and the Matrigel plug was removed, fixed with 3.7% formaldehyde/PBS and embedded in paraffin. The plugs were then sectioned and examined with Masson-Trichrome staining for microscopic observation. To quantify the formation of new blood vessel, the amount of hemoglobin (Hb) was measured using Drabkin reagent kit 525. The concentration of Hb was calculated from a known amount of Hb provided by the kit in parallel according to the supplier's protocol. The experiment was repeated 3 or 4 times, independently.

Absorption, Distribution and Excretion
Romidexin exhibits linear pharmacokinetic characteristics at standard doses.
44.5 L
8.4 L/h
Metabolism/Metabolites Romidexin is extensively metabolized in the liver primarily via CYP3A4 in vitro, with smaller contributions from CYP3A5, CYP1A1, CYP2B6, and CYP2C19.
Biological Half-Life Approximately 3 hours

Hepatotoxicity
In clinical trials of romidesin for the treatment of chronic advanced lung cancer (CTLC) and advanced-to-advanced lung cancer (PTLC), the incidence of elevated serum enzymes during treatment ranged from 7% to 20%, but these abnormalities were usually transient and mild, requiring no dose adjustment. 6% of patients experienced serum alanine aminotransferase (ALT) elevations exceeding 5 times the upper limit of normal (ULN). In pre-registration clinical trials of romidesin, no reports of hepatitis, jaundice, or clinically significant liver injury were observed in treated subjects. The clinical use of romidesin is limited, but there is no evidence that it is associated with significant liver injury. Romidesin also has immunomodulatory activity and has been reported to cause reactivation of latent DNA viruses, including Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and hepatitis B virus (HBV). One patient who was initially negative for hepatitis B surface antigen (HBsAg) but positive for hepatitis B core antibody (anti-HBc) and hepatitis B surface antibody (anti-HBs) subsequently experienced hepatitis B virus reactivation. However, the clinical manifestations of hepatitis B virus reactivation are usually mild, and it responds to oral antiviral therapy. In patients with Epstein-Barr virus (EBV)-associated lymphoid tissue (EBV-associated lymphoid tissue), romidesin is associated with severe reactivation of EBV infection and acute hepatitis, which can be severe or even fatal. Probability score: C (Possibly the cause of clinically significant liver damage, possibly due to reactivation of hepatitis B or EBV infection). High plasma protein binding (92%-94%).

[1]. Cancer Res. 2002 Sep 1;62(17):4916-21.

[2]. Br J Cancer. 2000 Sep;83(6):817-25.

[3]. Mol Cancer Ther. 2002 Sep;1(11):937-41.

[4]. Int J Cancer. 2002 Jan 20;97(3):290-6.

[5]. Biochem Pharmacol. 2002 Oct 1;64(7):1079-90.

[6]. Blood. 2003 Jul 15;102(2):652-8.

[7]. Cancer Res. 2006 Jul 15;66(14):7317-25.

[8]. Clin Cancer Res. 2008 Jan 15;14(2):549-58.

[9]. Clin Cancer Res. 2010 Jan 15;16(2):554-65.

Romidesin is a cyclic disulfide peptide composed of (2Z)-2-aminobutyric-2-enoyl, L-valine, (3S,4E)-3-hydroxy-7-thioheptane-4-enoyl, D-valine, and D-cysteine residues linked sequentially and cyclized end-to-end. It is an anti-tumor drug and an EC 3.5.1.98 (histone deacetylase) inhibitor. It is a cyclic disulfide peptide, an organic disulfide bond, and a heterocyclic antibiotic. Romidesin has been approved by the U.S. Food and Drug Administration (FDA) under the brand name Istodax for the treatment of certain types of cancer. Romidesin is currently being investigated as an investigational drug to explore strategies for curing HIV infection. As an investigational HIV drug, romidesin belongs to a class of drugs known as latency reversal agents. Romidesin is a selective histone deacetylase inhibitor approved in the United States in 2009 for the treatment of patients with cutaneous T-cell lymphoma (CTCL) who have received at least one prior systemic therapy. Romidesin's mechanism of action is as a histone deacetylase inhibitor, bile acid export pump inhibitor, organic anion transport peptide 1B1 inhibitor, organic anion transport peptide 1B3 inhibitor, organic anion transporter 1 inhibitor, and organic cation transporter 2 inhibitor. Romidesin is an intravenously administered histone deacetylase inhibitor and antitumor drug approved for the treatment of refractory or relapsed cutaneous and peripheral T-cell lymphomas. Romidesin may cause a slight increase in serum enzymes during treatment, but no clinically significant cases of liver injury have been found, although there are reports of hepatitis B virus reactivation.
Isothiocyanates (Istodax) have been reported in Humicola fuscoatra and Chromobacterium violaceum, with relevant data available.
Romidesin is a bicyclic peptide antibiotic isolated from Chromobacterium violaceum, possessing antitumor activity. Upon intracellular activation, romidesin binds to and inhibits histone deacetylase (HDAC), leading to altered gene expression and inducing cell differentiation, cell cycle arrest, and apoptosis. The drug also inhibits hypoxia-induced angiogenesis and depletes various heat shock protein 90 (Hsp90)-dependent oncoproteins.
Drug Indications
Romidesin is indicated for the treatment of adult cutaneous T-cell lymphoma (CTCL) in patients who have received at least one prior systemic therapy.
FDA Label
Treatment of peripheral T-cell lymphoma (PTCL),
Treatment of peripheral T-cell lymphoma (intranodal, other extranodal, and leukemia/disseminated)

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Mechanism of Action
Romidesin is a prodrug that is activated after entering the cell. Its active metabolite contains a free sulfhydryl group that interacts with zinc ions at the active sites of class 1 and class 2 HDAC enzymes, thereby inhibiting their enzyme activity. Some tumors exhibit HDAC overexpression and histone acetyltransferase downregulation/mutation. This imbalance between histone deacetylases (HDACs) and histone acetyltransferases leads to a reduction in regulatory genes, which in turn triggers tumorigenesis. Inhibition of HDACs may restore normal gene expression in cancer cells and lead to cell cycle arrest and apoptosis. FK228 is a histone deacetylase (HDAC) inhibitor, and the molecular mechanism of its inhibition is not yet clear. This study shows that the reduction of intramolecular disulfide bonds in FK228 significantly enhances its inhibitory activity, and the disulfide bonds can be rapidly reduced in cells through glutathione-mediated reduction. Computer models show that a thiol group of the reduced FK228 (redFK) interacts with the active site zinc, preventing substrate access. redFK has stronger inhibitory effects on HDAC1 and HDAC2 than on HDAC4 and HDAC6. Due to the rapid inactivation of redFK in culture medium and serum, its in vivo HDAC inhibitory activity is lower than that of FK228. Therefore, FK228, as a stable prodrug, can inhibit class I enzymes and is activated by reduction after being taken up by cells. Glutathione-mediated activation also suggests that it can be used clinically to combat glutathione-mediated resistance to chemotherapy. [1]

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Depsipeptide, FR901228, is a novel histone deacetylase cyclic peptide inhibitor with unique cytotoxic characteristics and is currently undergoing phase I clinical trials. This study demonstrates that FR901228, in addition to inducing G2/M phase arrest, also causes G1 phase arrest accompanied by Rb protein hypophosphorylation. In vitro kinase activity assays showed that FR901228 did not directly inhibit CDK activity, but an inhibitory effect was observed in CDK extracted from cells exposed to FR901228. Six to 12 hours after FR901228 treatment, cyclin D1 disappeared, while cyclin E was upregulated. Although FR901228 did not induce wild-type p53 expression, it could induce p21 (WAF1/CIP1) expression in a p53-independent manner. Cell clones lacking p21 did not arrest in G1 phase but continued DNA synthesis, and after FR901228 treatment, they arrested in G2/M phase. In addition, FR901228 inhibited EGF-induced ERK-2/MAPK activation, but since the overall tyrosine phosphorylation level of the cell was not affected after EGF stimulation, the early signal transduction events remained intact. Therefore, although FR901228 does not directly inhibit kinase activity, it leads to downregulation of cyclin D1 and p53-independent p21 induction, thereby inhibiting CDK activity and dephosphorylating Rb protein, ultimately causing cell growth to arrest in the early G1 phase. Unlike G1 phase arrest, G2/M phase arrest is not p21-dependent, but is associated with significant cytotoxicity. [2]

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Histone deacetylase inhibitors (HDIs) induce p21 expression at the transcriptional level by inhibiting histone deacetylation. We found that HDI sodium butyrate (Bu), trichostatin A (TSA), and depeptide (FR901228) all induced p21 expression, but only TSA and FR901228 induced mitotic arrest (and also G1 and G2 phase arrest). The ability to induce mitotic arrest was associated with the high cytotoxicity of these compounds. Despite inducing mitotic arrest, TSA and FR901228 (unlike paclitaxel) did not affect tubulin polymerization. Unlike FR901228, TSA induced acetylation of lysine residue 40 in tubulin; both soluble tubulin and microtubules were acetylated. p21 induction reached its maximum at 8 hours, while tubulin reached its maximum acetylation level 1 hour after TSA treatment. Tubulin acetylation was detected after treatment with 12-25 ng/ml TSA, but the acetylation level plateaued at 50 ng/ml TSA concentration, accompanied by G2/M phase arrest, the appearance of cells with DNA content below 2N, poly(ADP-ribose) polymerase cleavage, and rapid cell death. We concluded that histone deacetylase inhibitors (HDIs) have different effects on non-histone deacetylases, and TSA-induced rapid tubulin acetylation is a marker of the non-transcriptional effect of TSA. [3]

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FK228 (formerly FR901228) was recently isolated from Chromobacterium violaceum and is a potent antitumor drug whose target protein has been identified as histone deacetylase (HDAC). Due to its unique chemical structure (i.e., bicyclic peptide) and the activity characteristics shown in the National Cancer Institute (NCI) drug development program, FK228 is currently in the Phase I clinical trial stage for cancer treatment. This study investigated the anti-angiogenic activity of FK228 in vivo and in vitro. Results showed that, at the same concentration inhibiting histone deacetylase (HDAC) activity, FK228 effectively inhibited the proliferation, invasion, migration, adhesion, and tubular formation of hypoxic-stimulated bovine aortic endothelial cells. Furthermore, FK228 also inhibited angiogenesis in chicken embryos and adult mice in the Matrigel plug assay. Interestingly, FK228 inhibited the expression of angiogenesis-stimulating factors such as vascular endothelial growth factor or kinase insertion domain receptors, while inducing the expression of angiogenesis-inhibiting factors such as von Hippel-Lindau protein and neurofibrin 2, suggesting that FK228's inhibition of angiogenesis may involve gene transcription effects. These results indicate that FK228 is a novel anti-angiogenic drug that may at least partially inhibit tumor growth by suppressing angiogenesis. [4]

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FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tetracarben-3,6,9,19,22-pentanone; FR901228, depeptide] is a novel histone deacetylase inhibitor that has shown therapeutic efficacy in a phase I clinical trial for patients with malignant lymphoma. However, its mechanism of action remains unclear. This study investigated the in vitro and in vivo effects of FK228 on human lymphoma U-937 cells. FK228 exhibited a strong inhibitory effect on the growth of U-937 cells, with an IC50 value of 5.92 nM. In the SCID mouse lymphoma model, mice treated with FK228 once or twice weekly survived longer than control mice, with median survival of 30.5 days (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively (compared to 20 days in control mice). Notably, two of the 12 mice treated with FK228 (0.56 mg/kg, once or twice weekly) survived for more than 60 days of observation. Forty-eight hours after FK228 treatment, the apoptosis rate of U-937 cells increased to 37.7% over time. Furthermore, FK228 induced G1 and G2/M phase arrest in U-937 cells, leading to differentiation into the CD11b(+)/CD14(+) phenotype. Twenty-four hours after FK228 treatment, the expression of p21 (WAF1/Cip1) and gelosin mRNA increased by up to 654-fold and 152-fold, respectively. FK228 leads to histone acetylation of the p21 (WAF1/Cip1) promoter region (including the Sp1 binding site). In summary, (i) FK228 prolongs the survival of scid mice in a lymphoma model; (ii) the beneficial effects of FK228 on human lymphoma may be achieved through histone acetylation regulating gene expression, thereby inducing apoptosis, cell cycle arrest and differentiation. [5]

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Based on early observations, depsipeptide has been initiated in clinical trials to demonstrate its selective activity in chronic lymphocytic leukemia (CLL) in vitro. We aim to determine the relationship between histone H3 and H4 acetylation, histone deacetylase inhibition and apoptosis in CLL cells to validate the rationality of the pharmacodynamic endpoints in these clinical trials. We demonstrated that, in vitro, dicetilide at a concentration corresponding to the median lethal concentration (LC50, i.e., the concentration that causes 50% cell death) of cultured CLL cells (0.038 μM dicetilide) induces histone H3 and H4 acetylation and inhibits histone deacetylases. Changes in histone acetylation were lysine-specific, involving H4 K5, H4 K12, and H3 K9, and a lesser degree of H4 K8, but not H4 K16 or H3 K14. Depeptide-induced apoptosis was caspase-dependent, selectively activating the tumor necrosis factor (TNF) receptor (exogenous pathway), which in turn initiated the activation of caspase 8 and effector caspase 3. Activation of caspase 8 was accompanied by downregulation of the cellular FLICE repressor protein (c-FLIP, I-FLICE), but no upregulation of Fas (CD95) was observed. Changes in the expression of other apoptotic proteins, including Bcl-2, Bax, Mcl-1, and X-linked inhibitor of apoptosis (XIAP), were not observed. Our results suggest that inhibition of target enzyme histone deacetylases and histone H3 and H4 acetylation are associated with the TNF receptor-mediated apoptosis pathway, which is not utilized by other drugs for the treatment of chronic lymphocytic leukemia (CLL). These data suggest that histone H3 and H4 acetylation, histone deacetylase inhibition, and [6]

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the proteasome inhibitor bortezomib and the histone deacetylase inhibitor dixitide (FK228) upregulate tumor death receptors. Therefore, we investigated whether pretreatment of malignant cells with these drugs could enhance natural killer (NK) cell-mediated tumor killing. NK cells isolated from healthy donors and cancer patients were expanded in vitro and then their cytotoxicity to tumor cell lines was examined before and after exposure to bortezomib or dixitide. In 11 of 13 renal cell carcinoma lines (85%) and 16 of 37 other cancer cell lines (43%), exposure to these drugs significantly enhanced NK cell-mediated tumor lysis compared to untreated tumor controls (P < 0.001). Furthermore, NK cells pretreated with bortezomib or desipramide from metastatic renal cell carcinoma patients showed significantly enhanced cytotoxicity against autologous tumor cells compared to untreated tumors. Tumor cells sensitive to NK cell cytotoxicity showed significantly increased expression of DR5 (tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL)-R2; P < 0.05); conversely, the expression of MHC class I molecules, MIC-A/B, DR4 (TRAIL-R1), and Fas (CD95) remained unchanged. Blocking TRAIL on NK cells completely eliminated this enhanced NK cell killing sensitivity, while blocking DR5 on tumor cells partially eliminated it. These findings suggest that drug-induced TRAIL sensitivity could serve as a novel strategy to enhance the anticancer effects of adoptive NK cell infusion in cancer patients. [7]

C24H36N4O6S2

540.7

540.207

C, 53.31; H, 6.71; N, 10.36; O, 17.75; S, 11.86

128517-07-7

5352062

White to off-white solid powder

1.2±0.1 g/cm3

942.8±65.0 °C at 760 mmHg

219-224°C

524.0±34.3 °C

0.0±0.3 mmHg at 25°C

1.529

0.95

4

8

2

36

905

4

S1C([H])([H])[C@]2([H])C(N([H])/C(=C(/[H])\C([H])([H])[H])/C(N([H])C([H])(C(=O)O[C@]([H])(C([H])=C([H])C([H])([H])C([H])([H])S1)C([H])([H])C(N([H])[C@@]([H])(C(N2[H])=O)C([H])(C([H])([H])[H])C([H])([H])[H])=O)C([H])(C([H])([H])[H])C([H])([H])[H])=O)=O |c:27|

OHRURASPPZQGQM-GCCNXGTGSA-N

InChI=1S/C24H36N4O6S2/c1-6-16-21(30)28-20(14(4)5)24(33)34-15-9-7-8-10-35-36-12-17(22(31)25-16)26-23(32)19(13(2)3)27-18(29)11-15/h6-7,9,13-15,17,19-20H,8,10-12H2,1-5H3,(H,25,31)(H,26,32)(H,27,29)(H,28,30)/b9-7+,16-6-/t15-,17-,19-,20+/m1/s1

(1S,4S,7Z,10S,16E,21R)-7-ethylidene-4,21-di(propan-2-yl)-2-oxa-12,13-dithia-5,8,20,23-tetrazabicyclo[8.7.6]tricos-16-ene-3,6,9,19,22-pentone

NSC 630176;FK228; FK 228; Romidepsin; Chromadax; 128517-07-7; Antibiotic FR 901228; FK-228; FR901228; FR-901228; FR 901228; NSC-630176; NSC-630176; depsipeptide; US trade name: Istodax.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.85 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.08 mg/mL (3.85 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.85 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 1% DMSO+30% polyethylene glycol+1% Tween 80: 18mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)