Biomedical subjects

S K Kong

Publications and source records attributed to S K Kong.

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↗

PubMed2005

Real-time optical biosensor based on differential phase measurement of surface plasmon resonance.

We report a real-time differential phase measurement technique which can be implemented in optical surface plasmon resonance biosensors. The important feature of our design is that sensitivity has been greatly improved by measuring the differential phase change between the s and p-polarizations. Real-time measurement capability is achieved by using a phase extracting routine which continuously monitors the waveforms captured by two photo-detectors. Measurement capability of our setup is demonstrated through real-time monitoring of bovine serum albumin (BSA)/anti-BSA binding reaction. The estimated sensitivity of our current setup is 7.4 ng/ml.

Biosensing Techniques↗

PubMed2004

Highly sensitive differential phase-sensitive surface plasmon resonance biosensor based on the Mach-Zehnder configuration.

A high-sensitivity surface plasmon resonance (SPR) biosensor based on the Mach-Zehnder interferometer design is presented. The novel feature of the new design is the use of a Wollaston prism through which the phase quantities of the p and s polarizations are interrogated simultaneously. Since SPR affects only the p polarization, the signal due to the s polarization can be used as the reference. Consequently, the differential phase between the two polarizations allows us to eliminate all common-path phase noise while keeping the phase change caused by the SPR effect. Experimental results obtained from glycerin-water mixtures indicate that the sensitivity limit of our scheme is 5.5 x 10(-8) refractive-index units per 0.01 degrees phase change. To our knowledge, this is a significant improvement over previously obtained results when gold was used as the sensor surface. Such an improvement in the sensitivity limit should allow SPR biosensors to become a possible replacement for conventional biosensing techniques based on fluorescence. Monitoring of the bovine serum albumin (BSA) binding reaction with BSA antibodies is also demonstrated.

Antigen-Antibody Complex↗

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↗

PubMed2004

Chronic abdominal pain in a Chinese woman with spastic cerebral palsy.

Pain affects people's activities of daily living especially among those with cerebral palsy. We report a case of a woman with spastic cerebral palsy who presented with chronic abdominal pain. The principles of multidisciplinary pain management are highlighted and the difficulties when dealing with patients with special needs are discussed.

Abdominal Pain↗

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↗

PubMed2001

The nitroxide Tempol affords protection against ultraviolet radiation in a transgenic murine fibroblast culture model of cutaneous photoaging.

The generation of reactive oxygen species is among the various mechanisms by which ultraviolet radiation damages skin. Tempol, a superoxide dismutase analogue which readily penetrates cell membranes when administered exogenously, has been shown to provide protection against some forms of oxygen-dependent damage. In this study, we measured the ability of Tempol to protect against ultraviolet A- and ultraviolet B-induced damage, using a previously described transgenic mouse model of cutaneous photoaging. The ability of Tempol to prevent ultraviolet radiation-induced elastin promoter activation was determined in vitro. Tempol provided over 50% protection against ultraviolet B and over 70% protection against ultraviolet A as measured in our in vitro system. These results demonstrate the ability of the superoxide dismutase mimic, Tempol, to protect against ultraviolet induced elastin promoter activation. This compound could be a useful pharmacological agent to prevent cutaneous photoaging.

Animals↗

PubMed2001

Gliotoxin induces apoptosis in cultured macrophages via production of reactive oxygen species and cytochrome c release without mitochondrial depolarization.

The cytotoxicity and its underlying mechanisms induced by gliotoxin (GT), an immunosuppressive agent, in macrophages are poorly understood. We report here that GT induced a rapid apoptosis (DNA fragmentation and hypodiploid nuclei obtained within 4 hrs of treatment) in murine macrophages PU5-1.8 in a dose-dependent and cell cycle-independent manner. The GT-induced apoptosis was suppressed by z-Asp, z-VAD-fmk and antioxidants suggesting that production of reactive oxygen species (ROS) and activation of caspases were important in this process. Also, release of cytochrome c from mitochondria was found to be an early event (within 1 hr) after addition of GT (250 ng/ml) and its presence in the cytosol was sufficient to elicit apoptosis. Interestingly, the release of cytochrome c was not accompanied by a reduction in the mitochondrial membrane potential (psi m) as determined by several psi m-sensitive fluorescent indicators. Taken together, our results indicate that GT is a potent apoptotic agent in PU5-1.8 cells and the loss of psi m is not a universal early marker for apoptosis.

Animals↗

PubMed2001

cGMP abolishes agonist-induced [Ca(2+)](i) oscillations in human bladder epithelial cells.

Cytosolic calcium oscillations may permit cells to respond to information provided by increases in intracellular Ca(2+) concentration ([Ca(2+)](i) ) while avoiding prolonged exposure to constantly elevated [Ca(2+)](i). In this study, we demonstrated that agonists could induce Ca(2+) oscillations in human bladder epithelial cells. Application of 10 microM acetylcholine or 200 nM bradykinin triggered an initial Ca(2+) transient that was followed by periodic [Ca(2+)](i) oscillations. The oscillations did not depend on extracellular Ca(2+). 8-Bromoguanosine 3',5'-cyclic monophosphate abolished acetylcholine- or bradykinin-induced oscillations. Elevation of cellular cGMP by dipyridamole, an inhibitor of cGMP-specific phosphodiesterase, also terminated the [Ca(2+)](i) oscillations. The inhibitory effect of cGMP could be reversed by KT-5823, a highly specific inhibitor of protein kinase G (PKG), suggesting that the action of cGMP was mediated by PKG. Comparison of the effect of cGMP with that of xestospongin C, an inhibitor of the inositol 1,4,5-trisphosphate (IP(3)) receptor, revealed similarities between the action of cGMP and xestospongin C. Therefore, it is likely that cGMP and PKG may target a signal transduction step(s) linked to IP(3) receptor-mediated Ca(2+) release.

Acetylcholine↗

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

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

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c.

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypodiploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by alpha-D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.

Animals↗

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↗