Growth Inhibition of Human Gynecologic and Colon Cancer Cells by Phyllanthus watsonii through Apoptosis Induction

Phyllanthus watsonii Airy Shaw is an endemic plant found in Peninsular Malaysia. Although there are numerous reports on the anti cancer properties of other Phyllanthus species, published information on the cytotoxicity of P. watsonii are very limited. The present study was carried out with bioassay-guided fractionation approach to evaluate the cytotoxicity and apoptosis induction capability of the P. watsonii extracts and fractions on human gynecologic (SKOV-3 and Ca Ski) and colon (HT-29) cancer cells. P. watsonii extracts exhibited strong cytotoxicity on all the cancer cells studied with IC50 values of ≤ 20.0 µg/mL. Hexane extract of P. watsonii was further subjected to bioassay-guided fractionation and yielded 10 fractions (PW-1→PW-10). PW-4→PW-8 portrayed stronger cytotoxic activity and was further subjected to bioassay-guided fractionation and resulted with 8 sub-fractions (PPWH-1→PPWH-8). PPWH-7 possessed greatest cytotoxicity (IC50 values ranged from 0.66 – 0.83 µg/mL) and was selective on the cancer cells studied. LC-MS/MS analysis of PPWH-7 revealed the presence of ellagic acid, geranic acid, glochidone, betulin, phyllanthin and sterol glucoside. Marked morphological changes, ladder-like appearance of DNA and increment in caspase-3 activity indicating apoptosis were clearly observed in both human gynecologic and colon cancer cells treated with P. watsonii especially with PPWH-7. The study also indicated that P. watsonii extracts arrested cell cycle at different growth phases in SKOV-3, Ca Ski and HT-29 cells. Cytotoxic and apoptotic potential of the endemic P. watsonii was investigated for the first time by bioassay-guided approach. These results demonstrated that P. watsonii selectively inhibits the growth of SKOV-3, Ca Ski and HT-29 cells through apoptosis induction and cell cycle modulation. Hence, P. watsonii has the potential to be further exploited for the discovery and development of new anti cancer drugs

Expression of muscarinic acetylcholine receptors (M1-, M2-, M3- and M4-type) in the neuromuscular junction of the newborn and adult rat

Using intracellular recording and immunohistochemistry, we studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release in the neuromuscular junctions of the newborn (3-6 days postnatal) and adult (30-40 days) rat. In the Levator auris longus muscles of both newborn and adult rats, acetylcholine release was modified by the M1- receptor selective antagonists pirenzepine (10 µM) and MT-7 (100 nM) and by the M2-receptor selective antagonists methoctramine (1 µM) and AF-DX 116 (10 µM). The M4-receptor selective antagonists tropicamide (1 µM) and MT-3 (100 nM) can also modify the neurotransmitter release in certain synapses of the newborn muscles. The neurotransmitter release was not altered by the M3-receptor selective antagonist 4-DAMP (1 µM) in the adult or newborn rats. However, we directly demonstrate by immunocytochemistry the presence of these receptors in the motor endplates and conclude that M1-, M2-, M3- and M4-type muscarinic receptors are present in all the neuromuscular junctions of the rat muscle both in newborn and adult animals. These receptors may be located in the perisynaptic glial cell as well as at the nerve terminals

Topological differences along mammalian motor nerve terminals for spontaneous and alpha-Bungarotoxin-induced sprouting

Spontaneous sproutings can be observed in end plates from normal adult vertebrate muscles and motor end plates develop increased growth signs and sprouts when target muscle cells become less active or paralysed. Nevertheless, very little is known about where in the motor nerve terminal arborization spontaneous and experimentally induced sprouts originate, their similarities and differences and also about their final maturation or elimination. In this study we investigate the topological properties of both spontaneous and alpha-bungarotoxin-induced sprouts (during different periods of intoxication and after recovery) along the motor nerve terminal branches of the Levator auris longus muscle of Swiss mice (between 48- 169 day old). Muscles were processed for immunocytochemistry to simultaneously detect postsynaptic AChRs and axons. This procedurk permits us to make an accurate identification of the fine sprouts and a morphometric study of the presynaptic branching pattern profile in control muscles, during the toxin action and after recovery from paralysis. The results show that in normal muscles, the initial and trunk segments (those between branch points) of the terminal arborization sprouted proportionally more branches when taking their relative lengths into account than the distal free-end segments. In contrast, every micrometer of alpha-bungarotoxin-treated muscles throughout the full terminal arborization have the same probability of generating a sprout. Moreover, the toxininduced sprouts can consolidate as new branches once recovered from the paralysis without changing the total length of the nerve terminal arborization

Percutaneous Needle Electrolysis Reverses Neurographic Signs of Nerve Entrapment by Induced Fibrosis in Mice

Nerve entrapments such as carpal tunnel syndrome are the most common mononeuropathies. The lesional mechanism includes a scarring reaction that causes a vascular compromise. The most effective treatment is surgery, which consists of removing the scarred area, thus reverting the vascular impairment. In the present study, a more conservative therapeutic approach has been undertaken to release the nerve by means of galvanic current (GC) applied with a needle: percutaneous needle electrolysis (PNE). For this purpose, a mouse model of sciatic nerve entrapment has been created using albumin coagulated by glutaraldehyde (albumin 35% and glutaraldehyde 2% volume applied, 10 μl). After two weeks, a fibrous reaction was obtained which entrapped the nerve to the extent of causing atrophy of the leg musculature (14.7%, P<0.05 compared to the control leg). Ultrasound imaging confirmed that the model’s image was compatible with that of nerve entrapment in patients. To quantify the degree of entrapment, nerve conduction recordings were made. The amplitude (peak-to-peak) of the compound muscle action potential (CMAPs) decreased by 32.2% (P<0.05), and the proximal latency increases by 17.7% (P<0.05, in both cases). In order to release the sciatic nerve, PNE was applied (1.5 mA for 3 seconds and 3 repetitions; 1.5/3/3) by means of a solid needle in the immediacy of perineural fibrosis before and 5 minutes after the application of GC, and the proximal latency shows a decrease of 16% (P<0.05). The recovery of CMAPs amplitude was about 48.7% (P<0.05). Three weeks later, the CMAPs amplitude was almost completely recovered (94.64%). Therefore, with the application of GC by means of a solid needle, the sciatic nerve was definitively released from its fibrous entrapment

Chelating Langmuir-Blodgett film: a new versatile chemiluminescent sensing layer for biosensor applications

International audienceThe present study reports the achievement of a new chemiluminescent sensing layer able to simultaneously (i) play an active role on ligand immobilization and (ii) serve as a catalyst in detection processes for label-free biosensor applications. This new type of active Langmuir−Blodgett (LB) monolayer has been designed by using a chelating lipid (Ni-NTA-DOGS). Thanks to the chelated metallic cation, this peculiar lipid exhibits luminol chemiluminescence catalysis properties in the presence of hydrogen peroxide. Upon biomolecule interaction through imidazole ring chelation (mediated by the metallic cation bound to the lipid headgroup), the chemiluminescent signal can be modulated. The first chemiluminescent signal acquisition experiments have shown a strong and homogeneous signal of the chelating layer. Upon histamine interactions, a histidine derivative used as a marker of fresh food quality, we succeeded in obtaining as a proof of concept a chemiluminescent signal variation without any derivatization of the target molecule. This signal variation was shown to be directly correlated to the histamine concentration with a limit of detection of 2 μg/mL

Chelating Langmuir-Blodgett film: a new versatile chemiluminescent sensing layer for biosensor applications

International audienceThe present study reports the achievement of a new chemiluminescent sensing layer able to simultaneously (i) play an active role on ligand immobilization and (ii) serve as a catalyst in detection processes for label-free biosensor applications. This new type of active Langmuir−Blodgett (LB) monolayer has been designed by using a chelating lipid (Ni-NTA-DOGS). Thanks to the chelated metallic cation, this peculiar lipid exhibits luminol chemiluminescence catalysis properties in the presence of hydrogen peroxide. Upon biomolecule interaction through imidazole ring chelation (mediated by the metallic cation bound to the lipid headgroup), the chemiluminescent signal can be modulated. The first chemiluminescent signal acquisition experiments have shown a strong and homogeneous signal of the chelating layer. Upon histamine interactions, a histidine derivative used as a marker of fresh food quality, we succeeded in obtaining as a proof of concept a chemiluminescent signal variation without any derivatization of the target molecule. This signal variation was shown to be directly correlated to the histamine concentration with a limit of detection of 2 μg/mL