Sheng-Huei Yang

Up-regulation of Bax and endonuclease G, and down-modulation of Bcl-XL involved in cardiotoxin III-induced apoptosis in K562 cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III-induced K562 cell apoptosis was confirmed by DNA fragmentation (DNA ladder, sub-G1 formation) and phosphatidylserine (PS) externalization with an IC50 value of 1.7 µg/ml at 48 h. A mechanistic analysis demonstrated that CTX III-induced apoptotic cell death was accompanied by up-regulation of both Bax and endonuclease G (Endo G), and downregulation of Bcl-XL. CTX III had no effect on the levels of Bcl-2, Bid, XIAP survivin, and AIF proteins. CTX III treatment caused loss of the mitochondrial membrane potential (Δψm), release of mitochondrial cytochrome c to the cytosol, and activation of both caspase-9 and -3. CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor Z-VAD-FMK. However, CTX III did not generate reactive oxygen species (ROS) and antioxidants, including N-acetylcysteine and catalase, did not block CTX III-induced apoptosis in K562 cells. Modulation of Bax, Bcl-XL, and the Endo G proteins, release of mitochondrial cytochome c, and activation of caspase-3 and -9 all are involved in the CTX III-triggered apoptotic process in human leukemia K562 cells.

Cardiotoxin III induces apoptosis in K562 cells through a mitochondrial-mediated pathway.

1. Cardiotoxin (CTX) III is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III on human leukaemia K562 cells.

MECHANISMS OF CARDIOTOXIN III-INDUCED APOPTOSIS IN HUMAN COLORECTAL CANCER COLO205 CELLS

1 Cardiotoxin III (CTX III) is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III in human colorectal cancer Colo205 cells. 2 Cardiotoxin III‐induced Colo205 cell apoptosis was confirmed by DNA fragmentation (DNA ladder and sub‐G1 formation) with an IC50 of 4 mg/mL at 48 h. 3 Further mechanistic analysis demonstrate that CTX III induced the loss of mitochondrial membrane potential (Dym), cytochrome c release from mitochondria into the cytosol and activation of capase‐9, caspase 3, as well as markedly enhancing the expression of Bax, but not Bcl‐2, protein in the cells. Moreover, the CTX III‐induced apoptosis was significantly blocked by the broad‐spectrum caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone. 4 However, CTX III did not generate the formation of reactive oxygen species and anti‐oxidants, including N‐acetylcysteine, and catalase could not block CTX III‐induced apoptosis in the Colo205 cells. 5 Taken together, these results suggest that CTX III may induce apoptosis through a mitochondrial‐ and caspase‐dependent mechanism and alteration of Bax/Bcl‐2 ratio in human colorectal Colo205 cancer cells.

Involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in cardiotoxin III-induced apoptosis in HL-60 cells.

1 Cardiotoxin (CTX) III, a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. In the present study, we investigated the mechanisms underlying the anticancer activity of CTX III in human leukaemia (HL‐60 cells). 2 Cardiotoxin III activated the endoplasmic reticulum (ER) pathway of apoptosis in HL‐60 cells, as indicated by increased levels of calcium and glucose‐related protein 78 (Grp78), and triggered the subsequent activation of µ‐calpain and caspase 12. 3 In addition, CTX III initiated the mitochondrial apoptotic pathway in HL‐60 cells, as evidenced by an increased Bax/Bcl‐2 ratio, the release of cytochrome c and activation of caspase 9. 4 In the presence of 50 µmol/L Z‐ATAD‐FMK (a caspase 12 inhibitor) and 100 µmol/L Z‐LEHD‐FMK (a caspase 9 inhibitor), the CTX III‐mediated activation of caspase 9 and caspase 3 was significantly reduced. There was no significant effect of the caspase 12 inhibitor Z‐ATAD‐FMK on mitochondrial cytochrome c release. 5 Cardiotoxin III‐mediated activation of caspase 12 was not abrogated in the presence of the caspase 9 inhibitor Z‐LEHD‐FMK, indicating that caspase 12 activation was not downstream of caspase 9. 6 These results indicate that CTX III induces cell apoptosis via both ER stress and a mitochondrial death pathway.

Synthesis and antiproliferative evaluation of certain iminonaphtho[2,3-b]furan derivatives

Certain iminonaphtho[2,3-b]furan derivatives were synthesized from their respective carbonyl precursors in the regiospecific and the stereospecific manners. These compounds were evaluated for their antiproliferative effects against four human carcinoma cells (MCF7, NCI-H460, SF-268, and K562) and the normal fibroblast cell line (Detroit 551). Among them, (Z)-4-(hydroxyimino)naphtho[2,3-b]furan-9(4H)-one (8) and (Z)-4-methoxy-iminonaphtho[2,3-b]furan-9(4H)-one (9) exhibited GI(50) values of 0.82 and 0.60 microM, respectively, against the growth of K562 cells and were inactive against the normal fibroblast Detroit 551. The selectivity index (SI) on K562 cell for 8 and 9 was >121.95 and >166.67, respectively, which is comparable to daunorubicin (SI=239) and is more favorable than camptothecin (SI=16.5). The cell cycle analysis on K562 indicated that these compounds arrest the cell cycle at the G2/M phase. The morphological assessment and DNA fragmentation analysis indicated that 9-induced cell apoptosis in K562 cells. The apoptotic induction may through caspase-3 activity and cleavage of PARP.

A novel indoloquinoline derivative, IQDMA, induces S-phase arrest and apoptosis in promyelocytic leukemia HL-60 cells

N′-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1,2-diamine (IQDMA), an indoloquinoline compound, was identified in our laboratory as a novel antineoplastic agent with a broad spectrum of antitumor activity against many human cancer cells. Cell cycle analysis showed S-phase arrest and induction of apoptosis in HL-60 cells following 24 h exposure to IQDMA. Analysis of the cell cycle regulatory proteins demonstrated that IQDMA did not change the steady-state levels of cyclin B1, cyclin D3, and p21, but decreased the protein levels of Cdk1, Cdk2, and cyclin A. IQDMA also caused a marked increase in apoptosis, which was accompanied by increased levels of Bax, activated caspase-3, -8, and -9, and cleaved PARP. These molecular alterations provide an insight into IQDMA-caused growth inhibition, S-phase arrest, and apoptotic death of HL-60 cells.

Cardiotoxin III-induced apoptosis is mediated by Ca2+-dependent caspase-12 activation in K562 cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. When K562 cells were treated with CTX III, cytosolic calcium concentration was rapidly and persistently increased. This CTX III‐induced cell death was partially reversed by pretreatment with BAPTA/AM (20 µM), a chelator of intracellular Ca2+. Moreover, CTX III‐induced apoptotic signals, such as caspase‐12 and c‐Jun N‐terminal kinase (JNK) activation, were induced in a time‐dependent manner and inhibited by BAPTA/AM. In contrast, the neutral protease µ‐calpain, a key enzyme in endoplasmic reticulum (ER) stress‐related apoptosis via caspase‐12 activation, was unchanged during apoptosis. Taken together, our findings suggest CTX III‐induced apoptosis is triggered by Ca2+ influx, then activated caspase‐12 and JNK through µ‐calpain‐independent cascade, and consequently caused apoptosis.

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.

2-(6-Aryl-3(Z)-hexen-1,5-diynyl)anilines as a New Class of Potent Antitubulin Agents

Compounds 2a- h and 6 displayed significant GI 50 values of 10(-7)-10(-6) M against various cancer cell lines. Of these compounds, 2-(6-(2-trifluoromethylphenyl))-3(Z)-hexen-1,5-diynyl)aniline (2c) showed the most potent growth inhibition activity. Compound 2c also arrested cancer cells in the G2/M phase and in low concentration reduced a significant percentage of MDA-MB-231/ATCC breast cancer tetraploid cells. In addition to the G2/M block, compound 2c caused microtubule depolymerization and induced apoptosis via activation of the caspase family.

Synthesis and biological evaluation of novel symmetry bis-enediynes.

A series of acyclic symmetry bis-enediynes have been synthesized successfully and their bioactivities were evaluated. Among them, 1,6-bis(4-((2-(pyridin-2-ylethynyl)phenyl)ethynyl)phenoxy)hexane 8g showed good inhibition activity against the CCRF-CEM (GI(50)=0.04 microM) and HL-60 (GI(50)=0.09 microM) cell lines of human leukemia. The cell cycle analysis shows that compound 8g arrests cell cycle via inhibiting Cyclin A and Cyclin B expressions in low concentration and induces a significant apoptosis progress in high concentration.