Induction of apoptosis in SGC-7901 cells by iridium(III) complexes via endoplasmic reticulum stress-mitochondrial dysfunction pathway.

This study was intended to evaluate the anticancer activity of three newly synthesized iridium(III) complexes [Ir(ppy)2(PEIP)](PF6) (1) (ppy = 2-phenylpyridine, PEIP = 2-phenethyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(ppy)2(SIP)](PF6) (2) (SIP = (E)-2-styryl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(PEYIP)](PF6) (3) (PEYIP = 2-phenethynyl-1H-imidazo[4,5-f][1,10]phenanthroline). The cytotoxic activity in vitro against A549, SGC-7901, HepG2, HeLa and normal NIH3T3 cells was investigated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. We found that the complexes 1, 2 and 3 significantly inhibited cell proliferation, in particular, complexes 2 and 3 show high cytotoxic effect on SGC-7901 cells with an IC50 value of 5.8 ± 0.7 and 4.4 ± 0.1 µM. Moreover, cell cycle assay revealed that the complexes could block G2/M phase of the cell cycle. Apoptotic evaluation by Annexin V/PI staining indicated that complexes 1-3 can induce apoptosis in SGC-7901 cells. In addition, microscopy detection suggested that disruption of mitochondrial functions, characterized by increased generation of intracellular ROS and Ca2+ as well as decrease of mitochondrial membrane potential. Western blot analysis shows that the complexes upregulate the expression of pro-apoptotic Bax and downregulate the expression of anti-apoptotic Bcl-2, which further activates caspase-3 and prompts the cleavage of PARP. Taken together, these results demonstrated that complexes 1-3 exert a potent anticancer effect on SGC-7901 cells via ROS-mediated endoplasmic reticulum stress-mitochondrial apoptotic pathway and have a potential to be developed as novel chemotherapeutic agents for human gastric cancer. Three new iridium(III) complexes [Ir(ppy)2(PEIP)](PF6) (1) (ppy = 2-phenylpyridine, PEIP = 2-phenethyl-1H-imidazo[4,5-f][1,10]phenanthroline), [Ir(ppy)2(SIP)](PF6) (2) (SIP = 2-styryl-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)2(PEYIP)](PF6) (3) (PEYIP = 2-phenethynyl-1H-imidazo[4,5-f][1,10]phenanthroline) were synthesized and characterized. The anticancer activity in vitro was investigated by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The results show that the complexes induce apoptosis via ROS-mediated endoplasmic reticulum stress-mitochondrial dysfunction pathway.

Evaluation of anticancer effects in vitro of new iridium(III) complexes targeting the mitochondria.

Iridium(III) complexes have the potential to serve as novel therapeutic drugs for treating tumor. In this work, three new complexes [Ir(ppy)2(cdppz)](PF6) (1) (ppy = 2-phenylpyridine, cdppz = 11-chlorodipyrido[3,2-a,2',3'-c]phenazine), [Ir(bzq)2(cdppz)](PF6) (2) (bzq = benzo[h]quinolone) and [Ir(piq)2(cdppz)](PF6) (3) (piq = 1-phenylisoquinoline) were prepared as well as characterized. MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide) assay revealed that the complex 2 exerted potent cytotoxicity against to various cancer cells lines and particularly for SGC-7901 cells. Meanwhile, the complexes could suppress cell colonies formation and migration ability. Apoptosis assays of AO/EB staining as well as flow cytometry revealed that the synthesized complexes may cause apoptosis of SGC-7901 cells. Moreover, the decline of mitochondrial membrane potential (MMP), elevation of intracellular reactive oxygen species (ROS) levels and release of cytochrome c demonstrated the complexes could cause apoptosis mainly through the mitochondrial death pathway and arrest cell at G0/G1 phase. Additionally, the complexes have significant influence on the expression of proteins which is interrelated to cell apoptosis. In summary, our studies provided fundamental information regarding the further study of the possible anticancer mechanisms of iridium (III) complexes.

Liposome as drug delivery system enhance anticancer activity of iridium (III) complex.

Herein an Ir(III) complex [Ir(Hppy)2(HMNPIP)](PF6) (Ir1, Hppy = 2-phenylpyridine, HMNPIP = 2-(1H-imidazo[4,5-f][1, 10]phenanthroline-3-yl)-6-methoxy-4-nitrophenol) was prepared and characterized. Due to the low anticancer activity of Ir1 when administered free drug, we prepared a liposome Ir1Lipo encapsulated form of Ir1 to improve the antitumor effect, furthermore, we explored the antitumor mechanism of both forms in vitro experiments on HepG2 cells. We investigated the inhibitory efficiency of Ir1 and Ir1Lipo on cell viability and proliferation using MTT (MTT = 3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide) and colony-forming assay. Intracellular accumulation of reactive oxygen species (ROS) was examined using a fluorescence microscope (High Content Screening System, ImageXpress Micro XLS System, Molecular Devices LLC, Sunnyvale, CA), programmed cell death cells stained with acridine orange/ethidium bromide (AO/EB) using flow cytometry detection and western blot have been performed. An in vivo study where HepG2 cells were transplanted into nude nice as xenografts. Tumour volume and body weight were monitored during the 10 days of administration. After encapsulation in liposomes Ir1Lipo displayed high potency against a variety of tumour cells in vitro, especially against HepG2 (IC50 = 4.6 ± 0.5 µM). Mechanism studies indicated that Ir1Lipo initiated apoptosis by generating intracellular ROS that regulate lysosomal-mitochondrial dysfunction, followed by microtubule disruption that subsequently leads to a G0/G1 phase of cell cycle arrest. Additionally, Ir1Lipo significantly curbed tumour growth in nude mice. The tumour inhibitory rate was 51.2% (5.6 mg/kg). Therefore, liposome as a drug delivery system greatly enhances anticancer activity of Ir1 by a factor of relatively minor side effects.

The induction of apoptosis in SGC-7901 cells through the ROS-mediated mitochondrial dysfunction pathway by a Ir(III) complex.

A new ligand BTCP and its iridium(III) complex [Ir(ppy)2(BTCP)]PF6 (Ir-1) were synthesized and characterized by elemental analysis, ESI-MS, IR, 1H NMR and 13C NMR. The cytotoxic activity in vitro of the ligand and its complex against SGC-7901, HeLa, HOS, PC-12, BEL-7402, MG-63, SiHa, A549, HepG2 and normal cell LO2 were evaluated by MTT method [MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)]. The apoptosis was assayed with AO/EB and Hoechst 33258 staining methods. The reactive oxygen species (ROS), mitochondrial membrane potential, autophagy and cell invasion were studied under fluorescent microscope. The expression of caspases and Bcl-2 family proteins were investigated by western blot. The IC50 values of complex toward SGC-7901, BEL-7402 and MG-63 cells are 3.9 ± 0.5, 5.4 ± 1.2 and 4.2 ± 0.6 µM. The complex can increase the levels of ROS, and induce a decrease in the mitochondrial membrane potential. Ir-1 inhibits the cell growth at G0/G1 phase in SGC-7901 cells, and the complex can induce both autophagy and apoptosis and inhibit the cell invasion. And the complex induces apoptosis through a ROS-mediated mitochondrial dysfunction pathway.

Muscarinic activity modulated by C-type natriuretic peptide in gastric smooth muscles of guinea-pig stomach.

Natriuretic peptides (NPs) are a cyclic guanosine monophosphate (cGMP) generation system like nitric oxide (NO) and play an inhibitory regulation in gastrointestinal motility but the effect of NPs on muscarinic activity is still unclear. This study was designed to investigate effect of C-type natriuretic peptide (CNP) on muscarinic control of gastric motility and its ion channel mechanism. The spontaneous contraction of gastric smooth muscle strip was recorded by using physiograph in guinea-pig. Membrane currents and potential were recorded by using whole-cell patch-clamp technique. CNP significantly inhibited muscarinic M receptor agonist carbachol (Cch)-induced contractions of gastric smooth muscle strips and dramatically hyperpolarized Cch-induced depolarization of membrane potential in gastric single smooth muscle cell. Muscarinic currents induced by both Cch and GTPgammaS, a G-protein agonist were significantly suppressed by CNP. 8-Br-cGMP mimicked the effect of CNP on Cch-induced muscarinic currents, and the peak holding current was decreased from -200.66+/-54.35 pA of control to -67.35+/-24.82 pA. LY83583, a guanylate cyclase nonspecific inhibitor, significantly weakened the inhibitory effect of CNP on muscarinic current while zaprinast, a cGMP sensitive phosphoesterase inhibitor, potentiated the inhibitory effect of CNP on muscarinic current. cGMP production was dramatically enhanced by CNP and this effect was suppressed by LY83583 in gastric smooth muscle. These results suggest that CNP modulates muscarinic activity via CNP-NPR-particulate guanylate cyclase (pGC)-cGMP pathway in guinea-pig.

Effects of polyamines on contractility of guinea-pig gastric smooth muscle.

This study was designed to investigate the effects of polyamines on mechanical contraction and voltage-dependent calcium current (VDCC) of guinea-pig gastric smooth muscle. Mechanical contraction and calcium channel current I(Ba) were recorded by isometric tension recording and whole-cell patch clamp technique. Spermine, spermidine and putrescine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner. Spermine (2 mM) reduced high K+ (50 mM)-induced contraction to 16+/-6.4% of the control (n=9), and significantly inhibited I(Ba) in a reversible manner (p<0.05; IC50=0.8 mM). Pre- and post-treatment of tissue with spermine (2-5 mM, n=10) also inhibited acetylcholine (10 microM)-induced phasic contraction to 5+/-6.4% of the control. Inhibitory effect of spermine on I(Ba) was observed at a wide range of test potentials of current/voltage (I/V) relationship (p<0.05), and steady-state activation of I(Ba) was shifted to the right by spermine (p<0.05). Spermidine and putrescine (1 mM each) also inhibited I(Ba) to 51+/-5.7% and 81+/-5.3% of the control, respectively. And putrescine (1 mM) inhibited I(Ba) at whole tested potentials (p<0.05) without significant change of kinetics (p<0.05). Finally, 5 mM putrescine also inhibited high K+-induced contraction to 53+/-7.1% of the control (n=4). These findings suggest that polyamines inhibit contractions of guinea-pig gastric smooth muscle via inhibition of VDCC.

Relationship between atrial natriuretic peptide-immunoreactive cells and microvessels in rat gastric mucosa.

AIM: To investigate the ultrastructural localization of atrial natriuretic peptide (ANP)-synthesizing cells and the relationship between ANP-synthesizing cells and microvessels in rat gastric mucosa. METHODS: Immunohistochemistry techniques and postembedding immunoelectron microscopy techniques were used to validate the findings regarding the expression of ANP-synthesizing cells and the ultrastructural localization of ANP-synthesizing cells in the gastric mucosa. Histochemistry techniques and the tannic acid-ferric chloride method (TA-Fe staining method) were used to reveal microvessel density and the distribution of ANP-synthesizing cells in different regions of the stomach. RESULTS: Cells expressing ANP were localized and ANP-synthesizing cells were identified as enterochromaffin (EC) cells in the gastric mucosa. ANP-synthesizing cells existed in different regions of the stomach. The percentage ANP-synthesizing cells in the mucosa was greatest in the fundus (46.7%+/-5.3%), intermediate in the antrum (40.1%+/-4.5%), and least in the body (21.6%+/-3.6%). There was a positive relationship between the percentage of ANP-synthesizing cells and the density of microvessels in the antral mucosa, but not in the fundus or body mucosa. CONCLUSION: ANP is synthesized by EC cells in rat gastric mucosa, and ANP-synthesizing cells are most dense in the gastric fundus. ANP may act not only as a regional autocrine and/or paracrine regulator, but also as an endocrine regulatory peptide in the gastrointestinal tract.

Role of arachidonic acid in hyposmotic membrane stretch-induced increase in calcium-activated potassium currents in gastric myocytes.

AIM: To study effects of arachidonic acid (AA) and its metabolites on the hyposmotic membrane stretch-induced increase in calcium-activated potassium currents (I(KCa)) in gastric myocytes. METHODS: Membrane currents were recorded by using a conventional whole cell patch-clamp technique in gastric myocytes isolated with collagenase. RESULTS: Hyposmotic membrane stretch and AA increased both I(K(Ca))) and spontaneous transient outward currents significantly. Exogenous AA could potentiate the hyposmotic membrane stretch-induced increase in I(K(Ca)). The hyposmotic membrane stretch-induced increase in I(K(Ca)) was significantly suppressed by dimethyleicosadienoic acid (100 micromol/L in pipette solution), an inhibitor of phospholipase A2. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly suppressed AA and hyposmotic membrane stretch-induced increases in I(K(Ca)). External calcium-free or gadolinium chloride, a blocker of stretch-activated channels, blocked the AA-induced increase in I(K(Ca)) significantly, but it was not blocked by nicardipine, an L-type calcium channel blocker. Ryanodine, a calcium-induced calcium release agonist, completely blocked the AA-induced increase in I(K(Ca)); however, heparin, a potent inhibitor of inositol triphosphate receptor, did not block the AA-induced increase in I(K(Ca)). CONCLUSION: Hyposmotic membrane stretch may activate phospholipase A2, which hydrolyzes membrane phospholipids to ultimately produce AA; AA as a second messenger mediates Ca(2+) influx, which triggers Ca(2+)-induced Ca(2+) release and elicits activation of I(K(Ca)) in gastric antral circular myocytes of the guinea pig.