Ching-Ming Chien

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.

Naphtho[1,2-b]furan-4,5-dione induces apoptosis and S-phase arrest of MDA-MB-231 cells through JNK and ERK signaling activation.

Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits anti-carcinogenic effect. The results of present study showed that NFD inhibited the proliferation of breast cancer MDA-MB-231 cells through the induction of S-phase arrest and apoptosis. NFD-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclin A, cyclin B, and cyclin-dependent kinase (Cdk)2. NFD-induced apoptosis was characterized by increase of sub-G1 population, phosphatidylserine (PS) externalization, and activation of caspases. Moreover, up-regulation of Bad and down-regulation of Bcl-2, Bcl-X(L), and survivin led to the loss of mitochondrial membrane potential (DeltaPsim), the release of cytochrome c and sequential activation of caspase-9 and caspase-3. NFD activated c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK) in MDA-MB-231 cells. Inhibitors of JNK (SP600125) and ERK (PD98059), but not p38 MAPK (SB203580) suppressed NFD-induced S-phase arrest and apoptosis in MDA-MB-231 cells. Both SP600125 and PD98059 attenuated Bad up-regulation, and reversed down-regulation of Bcl-2, Bcl-X(L), survivin, cyclin A, cyclin B, and Cdk2 in NFD-treated cells. Taken together, our data show that JNK and ERK-signaling pathways play important roles in NFD-mediated S-phase arrest and apoptosis of MDA-MB-231 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.

Down-regulation of the JAK2/PI3K-mediated signaling activation is involved in Taiwan cobra cardiotoxin III-induced apoptosis of human breast MDA-MB-231 cancer 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. Exposure of MDA-MB-231 cells with 0.03, 0.09, and 0.15 microM of CTX III for 18 h, CTX III-induced cell apoptosis, as evidenced by accumulation of sub-G1 population, externalization of phosphatidylserine, loss of mitochondrial membrane potential (DeltaPsim) with subsequent release of cytochrome c, and activation of both capases-9 and caspase-3. This correlated with up-regulation in Bax and Bad, and down-regulation of various anti-apoptotic proteins, including Bcl-2, Bcl-X(L), and survivin in CTX III-treated cells. Mechanistic studies showed that CTX III suppressed the phosphorylation of JAK2, STAT3, Akt, and activation of PI3K. Moreover, the PI3K inhibitor wortmannin blocked activation of STAT3 and Akt without affecting the JAK2 activation, whereas JAK2 inhibitor AG490 suppressed the levels of phospho-STAT3, phospho-Akt, and PI3K, suggesting that PI3K activation occurs after JAK2 phosphorylation, and both PI3K and JAK2 kinases cooperate to mediate STAT3 and Akt phosphorylation. Both AG490 and wortmannin also led to up-regulation in Bax and Bad, and down-regulation of Bcl-2, Bcl-X(L), and survivin in MDA-MB-231 cells. Taken together, these results indicate that CTX III induces apoptosis in MDA-MB-231 cells via concomitant inactivation of the JAK2, STAT3, PI3K, and Akt signaling pathways.

Effects of Cardiotoxin III on NF-κB Function, Proliferation, and Apoptosis in Human Breast MCF-7 Cancer 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 apoptosis in human breast MCF-7 cancer cells was confirmed by sub-G1 formation, phosphatidylserine (PS) externalization, and poly (ADP-ribose) polymerase (PARP) cleavage with an IC50 of 2 microg/ml at 48 h. Effects of CTX III on proliferation and apoptosis correlated with upregulation of Bax, and downregulation of Bcl-XL, Bcl-2, and XIAP, with no appreciable alteration on the protein levels of Bid, Bim, and survivin. CTX III treatment also caused release of mitochondrial cytochrome c to the cytosol, which led to subsequent activation of capase-9. Moreover, CTX III inhibited the nuclear factor-kappaB (NF-kappaB) activation through inhibition of IkappaB kinase (IkappaK) activity. Overall, our results indicate that CTX III downregulates NF-kappaB in MCF-7 cells, leading to the suppression of proliferation and induction of apoptosis. These findings suggest the molecular basis for CTX III-induced apoptotic death of MCF-7 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.

Naphtho[1,2-b]furan-4,5-dione induces apoptosis of oral squamous cell carcinoma: involvement of EGF receptor/PI3K/Akt signaling pathway.

Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exerts an anti-tumor effect. This study was performed to elucidate whether the epidermal growth factor (EGF) receptor and phosphatidylinositol-3-kinase (PI3K) signaling pathways are involved in NFD-induced apoptosis of oral squamous cell carcinoma (OSCC). Immunoblot showed that NFD suppressed the phosphorylation of EGF receptor and activation of PI3K/Akt, downstream molecules of EGF receptor signaling pathway, in Ca9-22 cells. The levels of downstream targets of Akt, including phospho-glycogen synthase kinase-3beta (p-GSK-3beta), GSK-3beta, forkhead transcription factor (FKHR), and cyclin D1, were also reduced after NFD treatment. Moreover, inactivation of nuclear factor-kappaB (NF kappaB), modulation of I kappa K beta and I kappaB alpha, up-regulation of Bad, and down-regulation of anti-apoptotic proteins including phospho-Bad, Bcl-X(L), myeloid cell leukemia-1(Mcl-1), and XIAP were found in NFD-treated cells. In addition, NFD treatment disrupted mitochondrial membrane potential (Delta Psi m), resulted in release of cytochrome c, and activation of both caspases-9 and caspase-3. Taken together, these results indicate that NFD induces apoptosis in Ca9-22 cells via inactivation of the EGF receptor-mediated survival pathway.

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.

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.

Naphtho[1,2-b]furan-4,5-dione inactivates EGFR and PI3K/Akt signaling pathways in human lung adenocarcinoma A549 cells

AIMS Naphtho[1,2-b]furan-4,5-dione (NFD), prepared from 2-hydroxy-1,4-naphthoquinone and chloroacetaldehyde in an efficient one-pot reaction, exhibits an anti-carcinogenic effect. This study was performed to elucidate whether EGFR and PI3K signaling pathways are involved in NFD-induced apoptosis of human lung adenocarcinoma A549 cells. MAIN METHODS The effect of NFD on cell viability and apoptosis was measured by the MTT assay and flow cytometry. The phosphorylation levels of EGFR and its regulatory molecules by NFD treatment were studied by immunoblots. KEY FINDINGS Immunoblot showed that NFD inhibited EGFR phosphorylation and the activation of PI3K/Akt, downstream molecules of EGFR pathway, in A549 cells. The levels of downstream targets of Akt, including phospho-glycogen synthase kinase-3beta (p-GSK-3beta), GSK-3beta, forkhead transcription factor (FKHR), and cyclin D1, were also reduced after NFD treatment. Moreover, inactivation of nuclear factor-kappaB (NFkappaB), modulation of IkappaKalpha/beta and IkappaBalpha, up-regulation of Bad and Bax, and down-regulation of anti-apoptotic proteins including phospho-Bad, Bcl-2, survivin, and XIAP were also found in NFD-treated cells. In addition, NFD treatment disrupted mitochondrial membrane potential (DeltaPsim) and resulted in release of mitochondrial cytochrome c and activation of both caspases-9 and caspase-3. SIGNIFICANCE These findings indicate that EGFR and PI3K/Akt signaling pathways play important roles in NFD-induced apoptosis of A549 cells.