Tim-Tak Kwok

Reactive oxygen species mediate doxorubicin induced p53-independent apoptosis

Doxorubicin (DOX) is a common anticancer drug. The mechanisms of DOX induced apoptosis and the involvement of reactive oxygen species (ROS) in apoptotic signaling were investigated in p53-null human osteosarcoma Saos-2 cells. Accumulation of pre-G1 phase cells and induction of DNA laddering, which are the hallmarks of apoptosis, were detected in cells at 48 h upon DOX treatment. Furthermore, DOX increased the intracellular hydrogen peroxide and superoxide levels, followed by mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, prior to DNA laddering in Saos-2 cells. In addition, DOX treatment also upregulated Bax and downregulated Bcl-2 levels in the cells. The role of ROS in DOX induced cell death was confirmed by the suppression effect of catalase on DOX induced ROS formation, mitochondrial cytochrome c release, procaspase-3 cleavage, and apoptosis in Saos-2 cells. The catalase treatment however only suppressed DOX induced Bax upregulation but had no effect on Bcl-2 downregulation. Results from the present study suggested that ROS might act as the signal molecules for DOX induced cell death and the process is still functional even in the absence of p53.

Induction of apoptosis by green tea catechins in human prostate cancer DU145 cells

Green tea catechins (GTCs) including (-)-epigallocatechin-3-gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG) and (-)-epicatechin (EC) were shown to suppress cell growth and induce apoptosis in various cell systems in addition to their chemo-preventive effect. In this study, except EC which was inactive, green tea extract (TE) and other 3 GTCs were found to suppress the growth and induce apoptosis in human prostate cancer DU145 cells largely through an increase in reactive oxygen species formation and mitochondrial depolarization. The conclusion was supported by the fact that the profiles for different GTCs in growth suppression, apoptosis induction, ROS formation and mitochondrial depolarization are in a similar order, i.e. ECG > EGCG > EGC > EC. Although the molecular mechanisms are still not clear, apoptosis induced by GTCs is not related to the members of BCL-2 family as EGCG did not alter the expression of BCL-2, BCL-X(L) and BAD in DU145 cells.

Morin hydrate: a potential antioxidant in minimizing the free-radicals-mediated damage to cardiovascular cells by anti-tumor drugs.

The co-incubation of morin hydrate with either doxorubicin or mitomycin C could minimize the toxicity of these anti-tumor drugs on cardiovascular cells, such as red blood cells, human umbilical vein endothelial cells (ECV304) and primary mouse cardiomyocytes, whereas morin hydrate did not lower the cytotoxicity of the drugs on human hepatocellular carcinoma cells (HepG2). Morin hydrate may not exert its antioxidant effect by enhancing the antioxidant enzymatic activity because it did not cause any induction on the mRNA levels of manganese superoxide dismutase expression in ECV304 cells and HepG2 cells.

Mitochondrial targeting drug lonidamine triggered apoptosis in doxorubicin-resistant HepG2 cells

Mitochondria play a crucial role in the induction and execution of apoptosis. Accordingly, recent suggestions have been made to use agents that directly act on mitochondria to trigger apoptosis so that drug-sensitive and-resistant tumour cells can be eliminated. To test this hypothesis, human hepatocarcinoma HepG2 and its derivative R-HepG2 with doxorubicin (Dox) resistance as a result of expression of P-glycoprotein were used to investigate the effect of lonidamine (LND), a new mitochondrial targeting drug, on the induction of apoptosis. Results from our study indicate that R-HepG2 cells were more sensitive to LND than parental cells in terms of cytotoxicity determined by alamar blue assay. Cell death induced by LND was associated with the hallmarks of apoptosis such as mitochondrial membrane depolarization, release of cytochrome c, phosphatidyl-serine externalization and DNA fragmentation. Moreover, combined treatment of cells with Dox and LND elicited more cell death. Taken together, our results suggest a potential use of LND as an anti-cancer drug to bypass drug resistance and to trigger tumour destruction through apoptosis in HepG2 and R-HepG2 cells.

Estrogen-related receptor α (ERRα) inverse agonist XCT-790 induces cell death in chemotherapeutic resistant cancer cells

Estrogen-related receptor alpha (ERRalpha) is primarily thought to regulate energy homeostasis through interacting with peroxisome proliferator-activated receptor gamma coactivator-1alpha and -1beta (PGC-1alpha and -1beta). They coordinately control the transcription of genes in the oxidative phosphorylation pathway. In addition to its role in energy metabolism, ERRalpha has also been implicated as a prognostic marker for breast, ovarian, colon and prostate cancers. In this study, we found that an ERRalpha inverse agonist XCT-790 induced cell death in HepG2 hepatocarcinoma and its multi-drug resistance (MDR) sub-line R-HepG2. Using a dye Mitotracker Green which stains mitochondrion independent of mitochondrial membrane potential (DeltaPsi(m)), we found that XCT-790 dose-dependently decreased mitochondrial mass. Intriguingly, XCT-790 increased DeltaPsi(m) upon short term treatment but decreased DeltaPsi(m) upon longer term treatment. The changes of DeltaPsi(m) in turn promoted the production of reactive oxygen species (ROS) and led to ROS-mediated caspases 3/7, 8, 9 activation and cell death. Importantly, we established that an anti-oxidative compound Mn(III) Tetra(4-benzoic acid) porphyrin chloride (MnTBAP) blocked the caspases activities and cell death increased by XCT-790 treatment. Finally, we found that XCT-790 synergized with paclitaxel to induce cell death in multi-drug resistance sub-line R-HepG2. Our results provide a conceptual framework for further developing chemotherapeutics based on suppressing ERRalpha activity.

p53‐R175H mutant gains new function in regulation of doxorubicin‐induced apoptosis

Mutation of tumor suppressor p53 gene gains new function in regulation of DNA damage‐induced apoptotic response in tumor cells, which may lead to a poor response in cancer chemotherapy and radiotherapy. Transfection of mutant p53 (R175H) to p53‐null osteosarcoma Saos‐2 cells suppressed apoptosis induced by doxorubicin (DOX), cisplatin and gamma radiation. Downregulation of caspase‐3 but not ‐8 or ‐9 basal protein levels was also observed in Saos‐2 cells transfected with p53‐R175H. After 48 hr of DOX treatment, the rate of procasapse‐3 activation into 17 kDa active form was about 3‐fold higher in the control cells than that in the p53‐R175H counterpart. Gene silencing of p53‐R175H expression by p53 siRNA upregulate the procaspase‐3 protein level and restored DOX‐induced apoptosis in p53‐R175H cells. Our results suggest that p53‐R175H mutation may gain new function in decreasing DOX‐induced apoptotic response through suppression of caspase‐3 level and its activation.

Degree of pollution of several major streams entering Tolo Harbour, Hong Kong

Abstract Untreated domestic and industrial wastes are discharged into Tolo Harbour via several major streams entering the harbour. These untreated wastes will cause a harmful effect because Tolo Harbour is a land-locked sea with only a narrow outlet and an ever-decreasing volume caused by the construction of Plover Cove Reservoir and the reclamation of Shatin New Town. In order to study the effect of polluted water discharged into the harbour, water samples and sediments near the mouths of the major rivers were collected. The analysis of the total organic carbon, total nitrogen, and water soluble phosphate contents of the samples showed that Lam Tsuen River, Tai Po Kau River, and Shing Mon River were grossly polluted by organic and inorganic wastes. Blue Snake River which passes through the iron ore tailings accumulated high contents of metal contaminants.

Mitochondria-Targeting Drug Oligomycin Blocked P-Glycoprotein Activity and Triggered Apoptosis in Doxorubicin-Resistant HepG2 Cells

Background: Mitochondria are key regulators in apoptosis. This suggests that a mitochondrion can be a target for cancer treatment. To examine the feasibility of this approach, we investigated the effect of oligomycin on the induction of apoptosis in drug-resistant cells. As a mitochondrion-targeting agent, oligomycin inhibits mitochondrial F₀F1-ATPase. Of 37,000 molecules tested against the 60 human cancer cell lines of the National Cancer Institute, oligomycin is among the top 0.1% most cell line selective agents. Methods: Changes in the doxorubicin (Dox) accumulation and mitochondrial potential (Δψm) in human hepatocarcinoma HepG2 and its derivative R-HepG2 with Dox resistance were determined by flow cytometry. P-glycoprotein (Pgp) expression and release of cytochrome c from mitochondria were analyzed by Western blot. Cytotoxicity was examined by DNA fragmentation and the alamar blue assay. Results: R-HepG2 cells produced Pgp, showed drug resistance and accumulated less Dox when compared to their parent. In both cell lines, oligomycin depolarized Δψm, released cytochrome c and elicited DNA fragmentation. Moreover, oligomycin blocked Pgp activity and accumulated more Dox in R-HepG2. Combined treatment with Dox and oligomycin elicited more cell death. Conclusion: Our results suggest that oligomycin could bypass Dox resistance and trigger apoptosis in R-HepG2 cells.

The decrease of mitochondrial NADH dehydrogenase and drug induced apoptosis in doxorubicin resistant A431 cells

Doxorubicin (DOX) resistant A10A cells derived from human squamous carcinoma A431 cells were found to exhibit a smaller degree of apoptosis after DOX treatment as compared to their parent cells. Induction of reactive oxygen species (ROS) formation and mitochondrial depolarization by DOX were more pronounced in the parent cells than in the A10A cells. The fact that catalase suppressed the DOX effect on ROS induction, mitochondrial depolarization and apoptosis in both cell lines suggests an involvement of ROS in the DOX-induced apoptosis. To investigate the underlying mechanisms for DOX resistance in A10A cells, RT-PCR based differential display was used. One of the clones, which was down-regulated in the A10A cells, had sequence homology with part of the mitochondrial NADH dehydrogenase III (ND3) gene. NADH dehydrogenase plays an important role in generating ROS during DOX treatment. The results indicate that down-regulation of ND3 may at least in part contribute to the mechanism for A10A cells resistant to DOX-induced apoptosis.

Heavy metals inUlva lactuca collected within Tolo Harbour, an almost landlocked sea

SummaryThe sea lettuce (Ulva lactuca), a littoral and sublittoral marine macroalga grows abundantly around shorewaters of Hong Kong, especially Tolo Harbour whereU.lactuca is collected from the mudflat for local human consumption as well as for export. Samples ofU.lactuca, together with the ambient seawater and sediment, were collected from several localities around Tolo Harbour, an almost land-locked sea, receiving polluted water from several major streams. For comparison samples were also collected from areas near the unpolluted south coast of Hong Kong Island. It was demonstrated that the samples of seawater and sediment collected within Tolo Harbour were polluted from domestic, agricultural and industrial sources. The extent of the metal (copper, lead, zinc and iron) contamination was further reflected and amplified in the tissue ofU.lactuca.