Sensitization of TRAIL-resistant cervical cancer cells through combination of TRAIL and fucoxanthin treatments.

OBJECTIVE: Cervical cancer, the second most common cause of cancer death in women worldwide, is a malignant neoplasm arising from cells originating in the cervix uteri. Currently, surgery combined with chemo- and radiotherapy is the major therapeutic approach for women with early-stage cervical cancer. However, recurrent cervical cancers from acquired chemo-resistance remain a major cause of therapeutic failure. MATERIALS AND METHODS: In this study, we assessed the effects of the combination of TRAIL with fucoxanthin, which has been reported to suppress the cervical cancer cells growth on the cervical cancer treatments. HeLa cells, SiHa cells, and CaSki cells were used as in vitro model. Mice xenograft was used as in vivo model. TRAIL-resistant cells were generated from CaSki cell line. The activity of PI3K/Akt pathway was detected by Western blot. Cell viability was measured by MTT assay. RESULTS: We observed TRAIL-resistant cervical cancer cells were more sensitive to fucoxanthin treatments. By establishing a TRAIL-resistant cell line from CaSki, we found the TRAIL-resistant cells showed upregulated PI3K/Akt pathway. Moreover, CaSki TRAIL-resistant cells were more sensitive to the combination of TRAIL with either Akt inhibitor or fucoxanthin than treatment with TRAIL or fucoxanthin alone. Our in vitro and in vivo xenograft experiments demonstrate that the combination of TRAIL with fucoxanthin showed synergistically inhibitory effects on cervical cancer cells. CONCLUSIONS: The findings of this study suggest that the combined use of fucoxanthin and TRAIL might be a useful strategy against TRAIL-resistant cervical cancer.

In situ model for studying potassium currents in various growth plate chondrocyte subpopulations.

A new in situ model of partially digested growth plate cartilage suitable for patch clamp study of membrane currents of chondrocytes from various differentiation stages was developed. Thin sections of growth plate were enzyme digested to expose intact membranes of chondrocytes previously covered by extracellular matrix. This treatment dramatically increased the success rate of tight-seal formation from virtually 0% up to 40%. Whole-cell patch clamp recording revealed a delayed outward rectifying current as the major macroscopic current in chondrocytes of all differentiation stages. This current was sensitive to tetraethylammonium chloride and reversed polarity at a membrane potential close to the equilibrium potential of K+. Chondrocytes at resting stage expressed a much smaller K+ current than the proliferative and hypertrophic chondrocytes. When the current amplitudes were normalized for the cell membrane area, proliferative cells expressed a significantly higher outward current density.

Maturation of glutamatergic neurotransmission in dentate gyrus granule cells.

We studied the development of glutamatergic neurotransmission in dentate gyrus granule cells (GCs) in hippocampal slices from 5 to 12-day-old rats. The active postnatal neuronogenesis in dentate permits GCs with staggered birthdates to be studied in situ in a single preparation. We recorded evoked responses to medial perforant path stimulation using visually-guided whole-cell patch clamping to select immature GCs, and biocytin filling to correlate electrophysiologic responses with maturational stage. Even within this immature cell population we found four distinct electrophysiologic patterns. Type 1 cells had no glutamatergic current; Type 2 cells had only N-methyl-D-aspartate receptor (NMDA) current; Type 3 cells had both NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) current although the NMDA component could be isolated at low stimulus intensity (NMDA threshold/=AMPA threshold. Type 1 cells were least mature, and Type 4 cells most mature as assessed by cell properties, dendritic arborization, and penetration of dendrites into the molecular layer. Thus NMDA-mediated currents predominate early in GC development as is consistent with their role in processes that determine dentate architecture - neuronal migration, dendritic outgrowth and regression, and synapse stabilization. By analogy with 'silent synapses' (i.e. synapses that contain only NMDA receptors), Type 2 cells are candidate 'silent cells' that may undergo activity-dependent acquisition of functional fast-conducting AMPA receptors with maturation.

GABAA currents in immature dentate gyrus granule cells.

We used whole cell patch clamp and gramicidin perforated patch recordings in hippocampal slices to study gamma-aminobutyric acid (GABA) currents in granule cells (GCs) from juvenile rat dentate gyrus (DG). GCs are generated postnatally and asynchronously such that they can be detected at different stages of their maturation in DG within the first month. In contrast, inhibitory interneurons are generated embryonically, and their circuitry is well developed even as their target GCs and GC excitatory connections are still being formed. In this study, two GABA currents evoked in GCs by medial perforant path stimulation are compared. The first, pharmacologically isolated by glutamate receptor blockade, is the product of direct activation of GABA interneurons with monosynaptic input to the recorded GC (monosynaptic GABAA). Monosynaptic GABAA displays slight outward rectification of its current-voltage relation, is 97% eliminated by 10 microM bicuculline and coincides temporally with the excitatory components of GC postsynaptic currents as has been described for GABAA currents in other brain regions. The second is a novel GABA response that is detectable in 10 microM bicuculline and is present on GCs only at the earliest stages of their maturation. Unlike monosynaptic GABAA, this transient GABA is eliminated by glutamate receptor blockade and hence is likely to be generated by interneurons activated via an intervening glutamatergic synapse (polysynaptically). It is predominantly chloride mediated, has a relative bicarbonate/chloride permeability ratio of 26%, and is unchanged by bath-applied saclofen and strychnine or by intracellular calcium chelation. It is 97% antagonized by 100 microM picrotoxin and 99% antagonized by 100 microM bicuculline. This current is thus a relatively bicuculline (BMI)-resistant GABAA current (BMIR-GABAA). Compared with monosynaptic GABAA, BMIR-GABAA has a later peak, slower time course of decay, and marked outward rectification. Its reversal potential is 7-8 mV depolarized to that of monosynaptic GABAA whether recorded in whole cell or with gramicidin perforated patch to preserve native internal chloride concentration. Together these data may suggest that BMIR-GABAA is evoked by an anatomically segregated population of interneurons activating a unique, developmentally regulated GABAA receptor. Further, the transient nature of this current coupled with its temporal characteristics that preclude overlap with the excitatory components of the synaptic response are consistent with a role that is trophic or signaling rather than primarily inhibitory.

Pre-synaptic effect of the ATP-sensitive potassium channel opener diazoxide on rat substantia nigra pars reticulata neurons.

Spontaneous synaptic currents were recorded from visually identified substantia nigra pars reticulata (SNR) neurons in the rat brain slice preparation by whole-cell patch clamp technique. GABA neurons were distinguished from dopamine neurons by their electrophysiological characteristics. In the presence of 20 microM AP5 and CNQX, the spontaneous synaptic currents recorded from GABA neurons were sensitive to bicuculline and reversed polarity at a potential close to the equilibrium potential of Cl-, indicating that they were mediated by GABA(A) receptors. TTX at 1 microM eliminated action potential-dependent release of GABA from nerve terminals, revealing the miniature inhibitory post-synaptic currents (mIPSCs). The ATP-sensitive potassium channel (K(ATP) channel) opener diazoxide (30-300 microM) significantly reduced the frequency of the mIPSCs in a dose-dependent manner. However, diazoxide did not affect the average value and the distribution of the mIPSC amplitudes. Thus, this effect of diazoxide was pre-synaptic in nature. The K(ATP) channel blocker glibenclamide (300 microM) was able to restore the frequency of the mIPSCs. These data suggest that the striatonigral projection, which represents the major inhibitory input controlling SNR GABA neuron activities, possesses presynaptic K(ATP) channels on the nerve terminals.