m-3M3FBS

Phospholipase C activator m-3M3FBS protects against morbidity and mortality associated with sepsis.[Pubmed:22798676]

J Immunol. 2012 Aug 15;189(4):2000-5.

Although phospholipase C (PLC) is a crucial enzyme required for effective signal transduction and leukocyte activation, the role of PLC in polymicrobial sepsis remains unclear. In this study, we show that the direct PLC activator m-3M3FBS treatment significantly attenuates vital organ inflammation, widespread immune cell apoptosis, and mortality in a mouse sepsis model induced by lethal cecal ligation and puncture challenge. Mechanistically, m-3M3FBS-dependent protection was largely abolished by pretreatment of mice with the PLC-selective inhibitor U-73122, thus confirming PLC agonism by m-3M3FBS in vivo. PLC activation enhanced the bactericidal activity and hydrogen peroxide production of mouse neutrophils, and it also enhanced the production of IFN-gamma and IL-12 while inhibiting proseptic TNF-alpha and IL-1beta production in cecal ligation and puncture mice. In a second model of sepsis, PLC activation also inhibited the production of TNF-alpha and IL-1beta following systemic LPS challenge. In conclusion, we show that agonizing the central signal transducing enzyme PLC by m-3M3FBS can reverse the progression of toxic shock by triggering multiple protective downstream signaling pathways to maintain organ function, leukocyte survival, and to enhance microbial killing.

Effect of m-3m3FBS on Ca2+ handling and viability in OC2 human oral cancer cells.[Pubmed:22425810]

Acta Physiol Hung. 2012 Mar;99(1):74-86.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. m-3M3FBS at concentrations between 10-60 muM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. m-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 muM m-3M3FBS pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. At concentrations between 5 and 100 muM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 muM) induced apoptosis in a Ca2+-independent manner. Collectively, in OC2 cells, m-3M3FBS induced [Ca2+]i rise by causing inositol 1,4,5-trisphosphate-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive store-operated Ca2+ channels. m-3M3FBS also induced Ca2+-independent cell death and apoptosis.

M-3M3FBS-induced Ca(2) (+) movement and apoptosis in HA59T human hepatoma cells.[Pubmed:23347013]

Chin J Physiol. 2013 Feb 28;56(1):26-35.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca(2) (+) concentrations ([Ca(2) (+) ]i ) in HA59T human hepatoma cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca(2) (+) ]i levels in suspended cells by using fura-2 as a Ca(2) (+) -sensitive fluorescent dye. m-3M3FBS at concentrations of 10- 50 muM increased [Ca(2) (+) ]i in a concentration-dependent fashion. The Ca(2) (+) signal was reduced partly by removing extracellular Ca(2) (+) . m-3M3FBS-induced Ca(2) (+) influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca(2) (+) -free medium, 50 muM m-3M3FBS pretreatment inhibited the [Ca(2) (+) ]i rise induced by the endoplasmic reticulum Ca(2) (+) pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca(2) (+) ]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca(2) (+) ]i rise. At concentrations between 10 and 40 muM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca(2) (+) with 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis in a concentration-dependent manner. m-3M3FBS also increased levels of reactive oxygen species. Together, in human hepatoma cells, m-3M3FBS induced a [Ca(2) (+) ]i rise by inducing phospholipase C-independent Ca(2) (+) release from the endoplasmic reticulum and Ca(2) (+) entry via protein kinase C-sensitive store-operated Ca(2) (+) channels. m-3M3FBS induced cell death that might involve apoptosis via mitochondrial pathways.

Rise of [Ca(2)(+)]i and apoptosis induced by M-3M3FBS in SCM1 human gastric cancer cells.[Pubmed:24621336]

Chin J Physiol. 2014 Feb 28;57(1):31-40.

m-3M3FBS (2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide is a presumed phospholipase C activator which induced Ca(2)(+) movement and apoptosis in different cell models. How- ever, the effect of m-3M3FBS on cytosolic free Ca(2)(+) concentrations ([Ca(2)(+)]i) and apoptosis in SCM1 human gastric cancer cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca(2)(+)]i levels in suspended cells by using fura-2 as a Ca(2)(+)-sensitive fluorescent dye. m-3M3FBS at concentrations between 5-50 muM increased [Ca(2)(+)]i in a concentration-dependent manner. The Ca(2)(+) signal was reduced by half by removing extracellular Ca(2)(+). m-3M3FBS-induced Ca(2)(+) influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca(2)(+)-free medium, 50 muM m-3M3FBS pretreatment inhibited the [Ca(2)(+)]i rise induced by the endoplasmic reticulum Ca(2)(+) pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca(2)(+)]i rise. Suppression of inositol 1,4,5-trisphosphate production with U73122 did not change m-3M3FBS- induced [Ca(2)(+)]i rise. At concentrations between 25 and 50 muM m-3M3FBS killed cells in a concentration- dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca(2)(+) with acetoxy-methyl ester of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis at 25 and 50 muM. m-3M3FBS also increased levels of superoxide. Together, in human gastric cancer cells, m-3M3FBS induced a [Ca(2)(+)]i rise by inducing phospholipase C-independent Ca(2)(+) release from the endoplasmic reticulum and Ca(2)(+) entry via protein kinase C-sensitive store-operated Ca(2)(+) channels. m-3M3FBS induced cell death that might involve apoptosis via reactive oxygen species production.

The novel phospholipase C activator, m-3M3FBS, induces monocytic leukemia cell apoptosis.[Pubmed:15863272]

Cancer Lett. 2005 May 26;222(2):227-35.

We investigated the effect of the novel phospholipase C activator, m-3M3FBS, on the apoptosis of leukemic cells. m-3M3FBS inhibited the growth of the leukemic cell lines U937 and THP-1, but not primary monocytes. m-3M3FBS induced the apoptosis of U937 cells, which was accompanied by chromatin condensation and DNA fragmentation. Moreover, m-3M3FBS-induced apoptosis appeared to involve the down-regulation of anti-apoptotic Bcl-2, the up-regulation of pro-apoptotic Bax, the release of cytochrome c, and caspase activation. m-3M3FBS-induced apoptosis of U937 cells was also partly inhibited by BAPTA-AM and EGTA, indicating the involvement of intracellular calcium signaling on the apoptosis in U937 cells. The results of our study suggest that m-3M3FBS can be developed as a novel anti-leukemic agent.

Phospholipase C activator m-3M3FBS affects Ca2+ homeostasis independently of phospholipase C activation.[Pubmed:15302681]

Br J Pharmacol. 2004 Sep;143(1):3-7.

In this study, we have investigated responses to the phospholipase C (PLC) activator m-3M3FBS in SH-SY5Y human neuroblastoma cells. As measured using fura-2, m-3M3FBS caused a slowly developing - full response was obtained within 4-6 min - Ca(2+) elevation both in the presence and absence of extracellular Ca(2+), indicating Ca(2+) release from intracellular stores, putatively from endoplasmic reticulum and mitochondria. PLC activity was also measured using two methods, the classical ion-exchange separation and the more novel fluorescent real-time method. In the time frame in which m-3M3FBS caused Ca(2+) elevation (up to 7 min), no PLC activation was detected. Instead, more than 20 min were required to see any inositol phosphate generation in response to m-3M3FBS. m-3M3FBS also interfered with store-operated Ca(2+) influx and Ca(2+) extrusion. In conclusion, m-3M3FBS cannot be considered either potent or specific PLC activator.

Identification of a compound that directly stimulates phospholipase C activity.[Pubmed:12695532]

Mol Pharmacol. 2003 May;63(5):1043-50.

Phosphoinositide-specific phospholipase C (PLC) plays a pivotal role in the signal transduction of various cellular responses. However, although it is undeniably important that modulators of PLC activity be identified, no direct PLC activity modulator has been identified until now. In this study, by screening more than 10,000 different compounds in human neutrophils, we identified a compound that strongly enhances superoxide-generating activity, which is well known to be PLC-dependent. The active compound 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS) stimulated a transient intracellular calcium concentration ([Ca(2+)](i)) increase in neutrophils. Moreover, m-3M3FBS stimulated the formation of inositol phosphates in U937 cells, indicating that it stimulates PLC activity. The compound showed no cell-type specificity in terms of [Ca(2+)](i) increase in the various cell lines including leukocytes, fibroblasts, and neuronal cells. We also ruled out the possible involvement of heterotrimeric G proteins in m-3M3FBS-stimulated signaling by confirming the following: 1) pertussis toxin does not inhibit m-3M3FBS-induced [Ca(2+)](i) increase; 2) m-3M3FBS does not stimulate cyclic AMP generation; and 3) the inhibition of G(q) by the regulator of G protein-signaling 2 does not affect the m-3M3FBS-induced [Ca(2+)](i) increase. We also observed that m-3M3FBS stimulated PLC activity in vitro. The purified isoforms of PLC that were tested (i.e., beta2, beta3, gamma1, gamma2, and delta1) were activated by m-3M3FBS and showed no isoform specificity. Taken together, these results demonstrate that m-3M3FBS modulates neutrophil functions by directly activating PLC. Because m-3M3FBS is the first compound known to directly activate PLC, it should prove useful in the study of the basic molecular mechanisms of PLC activation and PLC-mediated cell signaling.