Biomedical subjects

T T Kwok

Publications and source records attributed to T T Kwok.

At least 19 recordsLinked to original sources

PubMed2006

The critical role of caspases activation in hypoxia/reoxygenation induced apoptosis.

Hypoxia/reoxygenation insult can be found in many tissues, including heart, brain, and tumor. It is believed that cell death may be resulted after cells were subjected to chronic hypoxia or reoxygenation after chronic hypoxia. The molecular mechanism for reoxygenation induced cell death is so far not clear and will require further study, in particular, to be distinguished from the pathways associated only with chronic hypoxia. In this study, the cell death mechanism in human squamous carcinoma A431 cells after hypoxia/reoxygenation insult is examined. It is demonstrated that although caspase-9 and -3 were activated during both hypoxia and reoxygenation, only those caspases activated during reoxygenation were responsible for reoxygenation induced apoptosis. Activation of caspase-9 and -3 during reoxygenation is believed to be triggered by the ROS formation at the time of reoxygenation. Addition of catalase during reoxygenation was found to attenuate reoxygenation induced apoptosis and caspase activation.

Apoptosis↗

PubMed2004

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 (Deltapsim) 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 Deltapsim, 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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

The role of Raf-1 in radiation resistance of human hepatocellular carcinoma Hep G2 cells.

Development of radiation resistance is one of the major reasons that cancer cells do not respond to radiotherapy and the mechanism for resistance is still not clear. Two sublines of human hepatocellular carcinoma Hep G2 cells were established from cells that survived two different irradiation regimes, 2 Gy for 10 days or 10 Gy for 2 days, respectively. Using MTT assay, the radiation conditioned cells were found to be more resistant to gamma-irradiation and have a greater extent of potentially lethal damage repair (PLDR) for radiation than the parent cells. By Western blot analysis, the radiation-conditioned cells were found to overexpress Raf-1 which is known to regulate the radiation resistance of cells. Inhibition of Raf-1 expression by antisense oligonucleotides increased the radiation sensitivity of the radiation-conditioned cells while inhibitors of Ras (L744,832), PI3K (LY294002) and p38 (SB203580) had no effect. Moreover, antisense Raf-1 oligonucleotides also decreased the radiation induced PLDR capacity of the radiation conditioned cells. It is therefore suggested that Raf-1 may induce radiation resistance through an increase in radiation induced PLDR capacity in Hep G2 cells.

Blotting, Western↗

PubMed2003

The nucleus of HeLa cells contains tubular structures for Ca2+ signaling with the involvement of mitochondria.

Calcium is an important messenger that controls many nuclear functions such as gene expression in mammalian cells but the regulation of nuclear Ca(2+) remains unclear. It has long been thought that Ca(2+) is translocated from the cytosol by a long distance to the nucleus through the nuclear pore complexes to activate or suppress gene transcription. However, this model is at best an incomplete one. With an aid of confocal and transmission electron microscopy, we demonstrated here that tubules, in a vertical or horizontal orientation, extended deep inside the nucleus of HeLa cells. These nuclear tubules (NTs) are double-membraned invaginations of the nuclear envelope and are usually associated with nucleolus. Also, membrane bound vesicles are found inside and inositol 1,4,5 trisphosphate (IP(3)) receptors are enriched in some but not all of these tubular structures. Interestingly, shuttling of mitochondria was observed in the NT and cytoplasm of the HeLa cells loaded with dihydro-rhod-2/AM. After stimulation with histamine that increases cytosolic [Ca(2+)] through IP(3) production, a slow rise of dihydro-rhod-2 fluorescence for the measurement of intra-mitochondrial Ca(2+) was observed in the area of NT indicating that Ca(2+) was sequestered by mitochondria inside the tubular invagination. Our work therefore suggests that the NTs and mitochondrial activities represent a specialized compartment and dynamic process involved in the regulation of Ca(2+) inside the cell nucleus.

Biological Transport↗

PubMed2003

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.

Antineoplastic Agents↗

PubMed2002

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.

Antineoplastic Agents↗

PubMed2001

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.

Anticarcinogenic Agents↗

PubMed2000

Reversal of TNF-alpha resistance by hyperthermia: role of mitochondria.

The aim of this study is to examine the effect of hyperthermia on tumour necrosis factor-alpha (TNF-alpha) resistance in L929-11E cells. L929-11E is a TNF-alpha resistant variant derived from L929 cells, a commonly used model for TNF-alpha study. Based on the results from flow cytometry and Western blotting, hyperthermia (43 degrees C, 3 h) was found to induce apoptosis, mitochondrial potential (delta psi(m)) depolarization and release of cytochrome c in L929-11E cells. Similar responses were found in L929 cells when treated with TNF-alpha. Heating at 43 degrees C for 1 h did not significantly damage the mitochondria of L929-11E cells but partially reversed their resistance to TNF-alpha. When L929-11E cells were sequentially treated with heating (43 degrees C, 1 h) and TNF-alpha, a more severe damage in mitochondria was observed. Taken together, our results indicate (1) hyperthermia induced apoptosis in L929-11E cells via mitochondrial damages in a way very similar to the action of TNF-alpha in L929 cells, (2) hyperthermia could be used to overcome TNF-alpha resistance by altering mitochondrial activities and (3) L929-11E and its parental cells provide a useful model in elucidating the signalling linkage between TNF-alpha receptor and mitochondria.

Cell Line↗

PubMed2000

Hyperthermia and tumour necrosis factor-alpha induced apoptosis via mitochondrial damage.

Hyperthermia is a potential anti-cancer regimen but the mode of action is far from clear. Based on the flow cytometric analysis with FITC-annexin V and propidium iodide, apoptosis was found to be the major form of cell death after the treatment with hyperthermia (43 degrees C, 3 h) and/or recombinant murine tumour necrosis factor-alpha (TNF-alpha, 50 ng/ml) in L929 cells. Since mitochondria are thought to play a key role in apoptosis, experiments were done to assess their role in the hyperthermia-mediated apoptosis. Our results indicate that hyperthermia was able to depolarize the mitochondrial membrane potential (delta psi m) and release cytochrome c to the cytoplasm, in a way very similar to the action of TNF-alpha. With the use of cyclosporin A to inhibit the delta psi m dissipation, the cytotoxicity mediated by hyperthermia or TNF-alpha was suppressed. Taken together, our results indicate that hyperthermia and TNF-alpha can induce apoptosis in L929 cells and the mitochondrial dysfunction plays a key role in the cell death process.

Animals↗

PubMed2000

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.

Animals↗

PubMed2000

The decrease of mitochondrial NADH dehydrogenease 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.

Antineoplastic Agents↗

PubMed2000

Slow rise of Ca2+ and slow release of reactive oxygen species are two cross-talked events important in tumour necrosis factor-alpha-mediated apoptosis.

Tumour necrosis factor-alpha (TNF-alpha) was found to be a cell cycle-independent apoptogenic cytokine in cultured fibroblast L929 cells. This assertion is based on the observations (1) TNF-alpha increased the number of cells with hypo-diploid DNA in a time dependent manner as revealed by flow cytometry, and (2) TNF-alpha induced DNA fragmentation as resolved by agarose gel electrophoresis. When cells were exposed to TNF-alpha (50 ng/ml), a slow rise in intracellular free Ca2+ level and a delayed increase in the production of reactive oxygen species (ROS) (both observed 3 h after the addition of TNF-alpha) were observed in fluo-3 and fura-red or dichlorofluorescein loaded cells, respectively. Interestingly, challenge of cells with TNF-alpha in the presence of BAPTA/AM, an intracellular Ca2+ chelator, decreased the release of ROS. Removal of ROS by 4-hydroxy 2,2,6,6-tetra-methyl-piperidinooxy (4OH-TEMPO) blocked the TNF-alpha-mediated Ca2+ rise. Moreover, when cells were exposed to TNF-alpha with both 4OH-TEMPO and BAPTA/AM, more viable cells were found than from treatment with either BAPTA/AM or 4OH-TEMPO. These results suggest that ROS and cellular Ca2+ are two cross-talk messengers important in TNF-alpha-mediated apoptosis.

Animals↗

PubMed1999

Enhanced cytotoxicity and suppression of glucose transport rate by combined treatment of recombinant human tumour necrosis factor-alpha and hyperthermia on L929 cells.

Combined treatment with human recombinant TNF-alpha (rhTNF-alpha) and hyperthermia at 43 degrees C arrested the growth of mouse fibrosarcoma L929 cells in vitro. The cytotoxic effect was enhanced in combined treatment compared with that following administration of rhTNF-alpha or hyperthermia alone. When the cells were subjected to hyperthermia at 43 degrees C for 3 hours and then incubated with 0.4 ng/ml rhTNF-alpha at 37 degrees C for 24 hours, a statistically significant 65% decrease in the rate of cellular glucose uptake was observed. This suppressive effect was synergistic in terms of effect achieved by rhTNF-alpha or hyperthermia individually. Since the growth of tumour cells depends mainly on catabolism of glucose, our findings indicate that one manner by which combined rhTNF-alpha and hyperthermia treatment inhibits L929 cell growth may be by reducing the supply of glucose to the cells.

Animals↗

PubMed1999

Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-alpha.

Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth and suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-alpha for 24 hours, the mRNA level of glucose transporter 1 (GLUT 1), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dose-dependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNF-alpha inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells.

Animals↗

PubMed1998

The nucleus of HeLa cell contains tubular structures for Ca2+ signalling.

It has long been assumed that Ca2+ are translocated from the cytosol to the cell nucleus by a long distance to activate transcription machinery buried deep in the nucleoplasm. However, this model has been recently challenged. When HeLa cells were loaded with fluo-3, highly fluorescent spots of approximately 2 microns in diameter were observed in the cell nucleus while the fluo-3 signals were low in their neighbouring nucleoplasm as determined by confocal microscopy. These fluorescent spots were devoid of but usually associated with chromatin on their boundary. When cells were stimulated by ionomycin (1 microM), the fluo-3 fluorescence in these spots increased faster than that in their neighbouring nucleoplasm. In another experiment, optical sections with hot spot(s) were used to construct 3-D images to study the morphology of the hot spots. Views of reconstruction from different angles indicated that the hot spots formed a tubular structure with a connection to the nucleocytoplasmic interface. Moreover, injection of calcium green-dextran (70 kDa), a Ca(2+)-sensitive indicator conjugated with an inert molecule of large molecular size, into the cytosol leads to a formation of signals also in a tubular shape inside the nucleoplasm. This suggests that the 'channels' are real inside the nucleus and they are derived from an invagination of the double-membraned nuclear envelope. Taken together, our results indicate (1) tubular structures are found inside the cell nucleus; (2) they are extended from the cytosol into the nucleus through the invagination of the double membraned nuclear envelope; (3) molecules of molecular size up to 70 kDa could penetrate into these 'tunnels'; (4) Ca2+ can be released or transported into the cell nucleus through these tubular structures after ionomycin stimulation; and (5) the structures are usually associated with chromatin.

Aniline Compounds↗

PubMed1997

Increases in mRNA levels of glucose transporters types 1 and 3 in Ehrlich ascites tumor cells during tumor development.

A common feature of many tumors is an increase in glucose catabolism during tumor growth. We studied the mechanism of this phenomenon by using Ehrlich ascites tumor bearing mice as the animal model. We found that Ehrlich ascites tumor cells possess only glucose transporter 1 (GLUT1) and GLUT3 but not GLUT2, GLUT4, or GLUT5. The mRNA levels of GLUT1 and GLUT3 increased progressively in the tumour during development; however, there were no changes observable in mRNA levels of glucose transporters of all types in brain, liver, and heart of the host mice. These findings suggest that Ehrlich ascites tumor augments its glucose transport mechanism relative to other tissues in response to its unique growth needs.

Animals↗

PubMed1995

Overexpression of the protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), in human epithelial ovarian cancer.

Our current understanding of human ovarian tumorigenesis is limited by the lack of a discrete precursor lesion as well as a limited knowledge of the steps in tumor progression. Since the alterations in the regulation of the tyrosyl residues on various cellular proteins appear to be an important pathway in neoplastic transformation, it is possible that changes in the expression of the proteins that control tyrosine phosphorylation (i.e., tyrosine kinases and phosphatases) may play a role in ovarian cancer development. Protein tyrosine phosphatase, nonreceptor type 6 (PTPN6), contains two src homology 2 domains and is expressed primarily in hematopoietic and epithelial cells. Using Northern blot and immunoblotting analysis, we showed that both the PTPN6 transcripts and proteins were overexpressed two- to four-fold in 7 of the 8 ovarian epithelial carcinoma cell lines studied. In addition, we showed that there was also a two- to threefold increase in expression of the PTPN6 transcript in 10 of 11 (91%) invasive ovarian epithelial cancer tissues examined. These observations suggest that the PTPN6 gene is potentially of etiologic relevance to a majority of ovarian cancers.

Epithelium↗

PubMed1994

Resistance to verapamil sensitization of multidrug-resistant cells grown as multicellular spheroids.

The ability of verapamil to overcome resistance to adriamycin in a multidrug-resistant derivative of the V79 cell line (LZ), grown as multicellular spheroids or as monolayers, was examined. Verapamil was much less effective in overcoming resistance to adriamycin in spheroids than in monolayers. Verapamil increased the adriamycin content of cells grown as monolayers, but had no significant effect on the drug content of spheroids. This occurred in spite of the same mdr-I mRNA and protein levels in monolayers and spheroids. When the surviving fraction of cells was normalized to the cellular adriamycin content, cells both in monolayers and spheroids treated with verapamil were still more sensitive to adriamycin than their counterparts not treated with verapamil. The observed resistance of spheroids to adriamycin and verapamil sensitization may be caused by a drug-resistance mechanism that is functional only in spheroids, in addition to the activity of P-glycoprotein. Multicellular tissue architecture and cell-cell contact may play significant roles in this type of multidrug-resistance mechanism.

Animals↗