MET Receptor Tyrosine Kinase Inhibition Reduces Interferon-Gamma (IFN-γ)-Stimulated PD-L1 Expression through the STAT3 Pathway in Melanoma Cells.

Melanoma is the leading cause of death from cutaneous malignancy. While targeted therapy and immunotherapy with checkpoint inhibitors have significantly decreased the mortality rate of this disease, advanced melanoma remains a therapeutic challenge. Here, we confirmed that interferon-gamma (IFN-γ)-induced PD-L1 expression in melanoma cell lines. This increased expression was down-regulated by the reduction in phosphorylated STAT3 signaling via MET tyrosine kinase inhibitor treatment. Furthermore, immunoprecipitation and confocal immunofluorescence microscopy analysis reveals MET and PD-L1 protein-protein interaction and colocalization on the cell surface membrane of melanoma cells. Together, these findings demonstrate that the IFN-γ-induced PD-L1 expression in melanoma cells is negatively regulated by MET inhibition through the JAK/STAT3 signaling pathway and establish the colocalization and interaction between an RTK and a checkpoint protein in melanoma cells.

Modulation of the PD-1/PD-L1 immune checkpoint axis during inflammation-associated lung tumorigenesis.

Chronic obstructive pulmonary disease (COPD) is a significant risk factor for lung cancer. One potential mechanism through which COPD contributes to lung cancer development could be through generation of an immunosuppressive microenvironment that allows tumor formation and progression. In this study, we compared the status of immune cells and immune checkpoint proteins in lung tumors induced by the tobacco smoke carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) or NNK + lipopolysaccharide (LPS), a model for COPD-associated lung tumors. Compared with NNK-induced lung tumors, NNK+LPS-induced lung tumors exhibited an immunosuppressive microenvironment characterized by higher relative abundances of PD-1+ tumor-associated macrophages, PD-L1+ tumor cells, PD-1+ CD4 and CD8 T lymphocytes and FOXP3+ CD4 and CD8 T lymphocytes. Also, these markers were more abundant in the tumor tissue than in the surrounding 'normal' lung tissue of NNK+LPS-induced lung tumors. PD-L1 expression in lung tumors was associated with IFNγ/STAT1/STAT3 signaling axis. In cell line models, PD-L1 expression was found to be significantly enhanced in phorbol-12-myristate 13-acetate activated THP-1 human monocytes (macrophages) treated with LPS or incubated in conditioned media (CM) generated by non-small cell lung cancer (NSCLC) cells. Similarly, when NSCLC cells were incubated in CM generated by activated THP-1 cells, PD-L1 expression was upregulated in EGFR- and ERK-dependent manner. Overall, our observations indicate that COPD-like chronic inflammation creates a favorable immunosuppressive microenvironment for tumor development and COPD-associated lung tumors might show a better response to immune checkpoint therapies.

Hyaluronan-CD44/RHAMM interaction-dependent cell proliferation and survival in lung cancer cells.

Although members of the hyaluronan (HA)-CD44/HA-mediated motility receptor (RHAMM) signaling pathway have been shown to be overexpressed in lung cancer, their role in lung tumorigenesis is unclear. In the present study, we first determined levels of HA and its receptors CD44 and RHAMM in human non-small cell lung cancer (NSCLC) cells and stromal cells as well as mouse lung tumors. Subsequently, we examined the role of HA-CD44/RHAMM signaling pathway in mediating the proliferation and survival of NSCLC cells and the cross-talk between NSCLC cells and normal human lung fibroblasts (NHLFs)/lung cancer-associated fibroblasts (LCAFs). The highest levels of HA and CD44 were observed in NHLFs/LCAFs followed by NSCLC cells, whereas THP-1 monocytes/macrophages showed negligible levels of both HA and CD44. Simultaneous silencing of HA synthase 2 (HAS2) and HAS3 or CD44 and RHAMM suppressed cell proliferation and survival as well as the EGFR/AKT/ERK signaling pathway. Exogenous HA partially rescued the defect in cell proliferation and survival. Moreover, conditioned media (CM) generated by NHLFs/LCAFs enhanced the proliferation of NSCLC cells in a HA-dependent manner as treatment of NHLFs and LCAFs with HAS2 siRNA, 4-methylumbelliferone, an inhibitor of HASs, LY2228820, an inhibitor of p38MAPK, or treatment of A549 cells with CD44 blocking antibody suppressed the effects of the CM. Upon incubation in CM generated by A549 cells or THP-1 macrophages, NHLFs/LCAFs secreted higher concentrations of HA. Overall, our findings indicate that targeting the HA-CD44/RHAMM signaling pathway could be a promising approach for the prevention and therapy of lung cancer.

Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart.

Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.

Adipocyte-Specific Deletion of Manganese Superoxide Dismutase Protects From Diet-Induced Obesity Through Increased Mitochondrial Uncoupling and Biogenesis.

Obesity and insulin resistance are associated with oxidative stress (OS). The causal role of adipose OS in the pathogenesis of these conditions is unknown. To address this issue, we generated mice with an adipocyte-selective deletion of manganese superoxide dismutase (MnSOD). When fed a high-fat diet (HFD), the AdSod2 knockout (KO) mice exhibited less adiposity, reduced adipocyte hypertrophy, and decreased circulating leptin. The resistance to diet-induced adiposity was the result of an increased metabolic rate and energy expenditure. Furthermore, palmitate oxidation was elevated in the white adipose tissue (WAT) and brown adipose tissue of AdSod2 KO mice fed an HFD, and the expression of key fatty acid oxidation genes was increased. To gain mechanistic insight into the increased fat oxidation in HFD-fed AdSod2 KO mice, we quantified the mitochondrial function and mitochondrial content in WAT and found that MnSOD deletion increased mitochondrial oxygen consumption and induced mitochondrial biogenesis. This effect was preserved in cultured adipocytes from AdSod2 KO mice in vitro. As expected from the enhanced fat oxidation, circulating levels of free fatty acids were reduced in the HFD-fed AdSod2 KO mice. Finally, HFD-fed AdSod2 KO mice were protected from hepatic steatosis, adipose tissue inflammation, and glucose and insulin intolerance. Taken together, these results demonstrate that MnSOD deletion in adipocytes triggered an adaptive stress response that activated mitochondrial biogenesis and enhanced mitochondrial fatty acid oxidation, thereby preventing diet-induced obesity and insulin resistance.

Combinatorial gene construct and non-viral delivery for anti-obesity in diet-induced obese mice.

The combinatorial peptidergic therapy of islet amyloid polypeptide (IAPP) and leptin (LEP) analogues was once an optimistic option in treating obese animals and patients. However, the need for frequent administrations and its negative side effects prevent it from being a viable choice. Here, we developed a combinatorial gene therapy of IAPP and LEP, where two genes are inserted into a single plasmid with self-cleaving furin and 2A sites to treat diet-induced obese (DIO) mice. The developed plasmid DNA (pDNA) individually produced both IAPP and LEP peptides in vitro and in vivo. The pDNA was delivered with a non-viral polymeric carrier, and its once-a-week administrations demonstrated a synergistic loss of body weight and significant reductions of fat mass, blood glucose, and lipid levels in DIO mice. The results suggest that the combinatorial gene therapy would have higher potential than the peptidergic approach for future translation due to its improved practicability.

UCP3 regulates cardiac efficiency and mitochondrial coupling in high fat-fed mice but not in leptin-deficient mice.

These studies investigate the role of uncoupling protein 3 (UCP3) in cardiac energy metabolism, cardiac O(2) consumption (MVO(2)), cardiac efficiency (CE), and mitochondrial uncoupling in high fat (HF)-fed or leptin-deficient mice. UCP3KO and wild-type (WT) mice were fed normal chow or HF diets for 10 weeks. Substrate utilization rates, MVO(2), CE, and mitochondrial uncoupling were measured in perfused working hearts and saponin-permeabilized cardiac fibers, respectively. Similar analyses were performed in hearts of ob/ob mice lacking UCP3 (U3OB mice). HF increased cardiac UCP3 protein. However, fatty acid (FA) oxidation rates were similarly increased by HF diet in WT and UCP3KO mice. By contrast, MVO(2) increased in WT, but not in UCP3KO with HF, leading to increased CE in UCP3KO mice. Consistent with increased CE, mitochondrial coupling was increased in the hearts of HF-fed UCP3KO mice. Unexpectedly, UCP3 deletion in ob/ob mice reduced FA oxidation but had no effect on MVO(2) or CE. In addition, FA-induced mitochondrial uncoupling was similarly enhanced in U3OB compared with ob/ob hearts and was associated with elevated mitochondrial thioesterase-1 protein content. These studies show that although UCP3 may mediate mitochondrial uncoupling and reduced CE after HF feeding, it does not mediate uncoupling in leptin-deficient states.

Early mitochondrial adaptations in skeletal muscle to diet-induced obesity are strain dependent and determine oxidative stress and energy expenditure but not insulin sensitivity.

This study sought to elucidate the relationship between skeletal muscle mitochondrial dysfunction, oxidative stress, and insulin resistance in two mouse models with differential susceptibility to diet-induced obesity. We examined the time course of mitochondrial dysfunction and insulin resistance in obesity-prone C57B and obesity-resistant FVB mouse strains in response to high-fat feeding. After 5 wk, impaired insulin-mediated glucose uptake in skeletal muscle developed in both strains in the absence of any impairment in proximal insulin signaling. Impaired mitochondrial oxidative capacity preceded the development of insulin resistant glucose uptake in C57B mice in concert with increased oxidative stress in skeletal muscle. By contrast, mitochondrial uncoupling in FVB mice, which prevented oxidative stress and increased energy expenditure, did not prevent insulin resistant glucose uptake in skeletal muscle. Preventing oxidative stress in C57B mice treated systemically with an antioxidant normalized skeletal muscle mitochondrial function but failed to normalize glucose tolerance and insulin sensitivity. Furthermore, high fat-fed uncoupling protein 3 knockout mice developed increased oxidative stress that did not worsen glucose tolerance. In the evolution of diet-induced obesity and insulin resistance, initial but divergent strain-dependent mitochondrial adaptations modulate oxidative stress and energy expenditure without influencing the onset of impaired insulin-mediated glucose uptake.

Tempol inhibits growth of As4.1 juxtaglomerular cells via cell cycle arrest and apoptosis.

A stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-osyl (Tempol) is widely used as an antioxidant in vitro and in vivo. In this study, we investigated the effects of Tempol on the growth of As4.1 juxtaglomerular cells in relation to cell cycle and cell death. Tempol dose-dependently decreased the growth of As4.1 cells with an IC50 of ~1 mM at 48 h. DNA flow cytometry analysis and BrdU staining indicated that Tempol induced S phase arrest, which is accompanied by a downregulation of cyclin A. Tempol also induced apoptotic cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ∆Ψm), an activation of caspase-3 and cleavage of poly(ADP-ribose)polymerase-1 (PARP-1). Furthermore, Tempol increased reactive oxygen species (ROS) levels, and the phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). MEK and JNK inhibitors significantly attenuated a growth inhibition in Tempol-treated As4.1 cells. In conclusion, Tempol inhibited the growth of As4.1 cells via cell cycle arrest and apoptosis. Tempol also activated ERK and JNK signaling, which was responsible for cell growth inhibition. Our present data provide useful information for the toxicological effects of Tempol in juxtaglomerular cells in relation to cell growth inhibition and cell death.

Intracellular glutathione levels are involved in carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone-induced apoptosis in As4.1 juxtaglomerular cells.

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. In the present study, we investigated the involvement of reactive oxygen species (ROS) and glutathione (GSH) in FCCP-induced As4.1 juxtaglomerular cell death. Intracellular ROS levels were decreased by FCCP at the early time points (10-150 min) and increased at 48 h. FCCP inhibited the activity of Mn-superoxide dismutase (Mn-SOD) via down-regulating its protein expression. Ebselen (an antioxidant) significantly attenuated ROS levels in FCCP-treated cells, but did not prevent FCCP-induced cell death. Moreover, intracellular GSH content was rapidly diminished within 10 min of FCCP treatment, which was accompanied by a reduction of the mitochondrial membrane potential [MMP (∆ψm)]. L-buthionine sulfoximine (BSO, a GSH synthesis inhibitor) significantly augmented As4.1 cell death by FCCP. However, N-acetylcysteine (NAC, a GSH precursor and antioxidant) attenuated GSH depletion, MMP (∆ψm) loss and cell death in FCCP-treated As4.1 cells. In addition, NAC increased Mn-SOD activity and decreased ROS levels in FCCP-treated As4.1 cells. In conclusion, these results suggest that compared to ROS levels, intracellular GSH levels are more closely linked to FCCP-induced apoptosis in As4.1 juxtaglomerular cells.

Carbonyl cyanide p-(trifluoromethoxy) phenylhydroazone induces caspase-independent apoptosis in As4.1 juxtaglomerular cells.

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in mitochondria. This study evaluated the effects of FCCP on the growth of juxtaglomerular As4.1 cells in relation to the cell cycle and apoptosis. FCCP inhibited the growth of As4.1 cells with an IC(50) of approximately 10 muM at 48 hours. DNA flow cytometry indicated that FCCP did not induce the specific phase arrests of the cell cycle. This agent efficiently reduced mitochondrial membrane potential (MMP; DeltaPsi(m)) levels within 1 hour and dose-dependently induced loss of MMP (DeltaPsi(m)) at 48 h. FCCP also induced apoptosis in As4.1 cells, as evidenced by sub-G(1) cells and annexin V binding assay. This apoptotic process was accompanied by caspase-3 activation and PARP cleavage. Although caspase-3 inhibitor significantly reduced the activity of caspase-3, all of the caspase inhibitors tested in this study failed to rescue As4.1 cells from FCCP-induced cell death. In conclusion, this study demonstrated that FCCP, as a mitochondria-damaging agent, potently induces apoptosis in As4.1 juxtaglomerular cells via a caspase-independent manner.

Attenuation of MG132-induced HeLa cell death by N-acetyl cysteine via reducing reactive oxygen species and preventing glutathione depletion.

MG132 as a proteasome inhibitor can induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, the effects of N-acetyl cysteine (NAC; an antioxidant) on MG132-induced HeLa cell death in relation to ROS and glutathione (GSH) were investigated. MG132 induced cell growth inhibition and apoptosis in HeLa cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; Delta Psi(m)), activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage. MG132 increased ROS levels, including O(2)(*-), and GSH depleted cell numbers of HeLa cells. NAC reduced the number of annexin V-positive cells and MMP (Delta Psi(m)) loss by MG132. In addition, NAC significantly reduced the ROS level and prevented GSH depletion. In conclusion, NAC prevented MG132-induced HeLa cell death via decreasing ROS and preventing GSH depletion.

Treatment with p38 inhibitor intensifies the death of MG132-treated As4.1 juxtaglomerular cells via the enhancement of GSH depletion.

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MG132 and/or MAPK inhibitors on As4.1 juxtaglomerular cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. MG132 inhibited the growth of As4.1 cells and induced cell death, accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)) and activation of caspase-3 and -8. MG132 increased ROS levels, and GSH depleted cell numbers. The MEK inhibitor slightly reduced cell growth and caspase-3 activity in MG132-treated As4.1 cells and mildly increased MMP (DeltaPsi(m)) loss and O(2)(*-) level. However, it did not increase apoptosis and GSH depletion. The JNK inhibitor did not strongly influence cell growth, cell death, and GSH depletion by MG132, but increased caspase-3 activity, MMP (DeltaPsi(m)) loss, and O(2)(*-) level. Treatment with the p38 inhibitor magnified cell-growth inhibition and apoptosis by MG132. This agent also strongly increased caspase-8 activity, MMP (DeltaPsi(m)) loss, O(2)(*-) level, and GSH depletion. Conclusively, the p38 inhibitor strongly intensified cell death in MG132-treated As4.1 cells. The changes of GSH content by MG132 and/or MAPK inhibitors were closely related to the death of As4.1 cells.

Proteasome inhibitor MG132 reduces growth of As4.1 juxtaglomerular cells via caspase-independent apoptosis.

The proteasome inhibitor MG132 has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). Here, we investigated the molecular mechanisms of MG132 in As4.1 juxtaglomerular cell death in relation to apoptosis and levels of ROS and glutathione (GSH). MG132 inhibited the growth of As4.1 cells with an IC(50) of approximately 0.3-0.4 microM at 48 h and induced cell death, accompanied by the loss of mitochondrial membrane potential (MMP; Psi(m)), Bcl-2 decrease, activations of caspase-3 and caspase-8, and PARP cleavage. MG132 increased intracellular ROS levels and GSH-depleted cell numbers. However, caspase inhibitors, especially Z-VAD (pan-caspase inhibitor) intensified cell growth inhibition, cell death, MMP (Psi(m)) loss, and Bcl-2 decrease in MG132-treated As4.1 cells. Z-VAD also slightly intensified increases in ROS levels and GSH depletion in MG132-treated As4.1 cells. In conclusion, MG132 reduced the growth of As4.1 cells via caspase-independent apoptosis. The changes in ROS and GSH levels by MG132 and caspase inhibitors partially influenced the growth inhibition and death of As4.1 cells.

Treatment with p38 inhibitor partially prevents Calu-6 lung cancer cell death by a proteasome inhibitor, MG132.

MG132 (carbobenzoxy-Leu-Leu-leucinal) as a proteasome inhibitor has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, we investigated the effects of MEK (mitogen-activated protein [MAP] kinase or extracellular signal-regulated kinase [ERK] kinase) or p38 inhibitor on MG132-treated Calu-6 lung cancer cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. Treatment with 10 mumol/L MG132 inhibited the growth of Calu-6 cells at 24 hours. MG132 induced apoptosis in Calu-6 cells, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)). ROS were increased in MG132-treated Calu-6 cells. MG132 also induced GSH depletion in Calu-6 cells. Treatment with MEK inhibitor did not significantly affect cell growth, cell death, ROS, and GSH levels in MG132-treated Calu-6 cells. Furthermore, MG132 increased the phosphorylation of p38 in Calu-6 cells at 1 and 24 hours. Treatment with p38 inhibitor significantly prevented cell growth inhibition, MMP (DeltaPsi(m)) loss and apoptosis in MG132-treated Calu-6 cells. This inhibitor increased ROS level and decreased GSH depletion in these cells. In conclusion, p38 inhibitor partially prevented Calu-6 cell death by MG132, which might be affected by GSH level changes.

MG132, a proteasome inhibitor, induced death of calf pulmonary artery endothelial cells via caspase-dependent apoptosis and GSH depletion.

MG132, a proteasome inhibitor, has been shown to induce apoptotic cell death through formation of reactive oxygen species (ROS). Here, we evaluated the effects of MG132 on the growth of endothelial cells, especially calf pulmonary artery endothelial cells (CPAECs). MG132 dose-dependently inhibited the growth of CPAECSs and human umbilical vein endothelial cells (HUVECs) at 24 hours. MG132 also induced apoptosis in both cell lines, which was accompanied by the loss of mitochondrial membrane potential. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 and -9 inhibitor) significantly rescued CPAECs from MG132-induced cell death. MG132 increased ROS level and GSH depleted cell numbers of CPAECs. None of the caspase inhibitors reduced ROS level in MG132-treated CPAECs but did reduce apoptosis in these cells. In conclusion, MG132 inhibited the growth of endothelial cells, especially CPAECs via caspase-dependent apoptosis. MG132-induced CPAEC death was related to GSH depletion rather than a change in ROS level.

Propyl gallate inhibits the growth of endothelial cells, especially calf pulmonary arterial endothelial cells via caspase-independent apoptosis.

Propyl gallate (PG) as a synthetic antioxidant exerting a variety of effects on tissue and cell functions. We evaluated the effects of PG on the growth of endothelial cells, especially calf pulmonary artery endothelial cells (CPAEC) in relation to apoptosis. PG dose-dependently inhibited the growth of CPAEC and human umbilical vein endothelial cells (HUVEC) at 24 h. The susceptibility of CPAEC to PG was higher than that of HUVEC. PG induced apoptosis in CPAEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsim). The tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) did not rescue CPAEC from PG-induced cell death but instead slightly enhanced the cell death. PG increased reactive oxygen species (ROS) level in CPAEC. The caspase inhibitors did not significantly change the ROS level. Furthermore, PG increased the GSH depleted cell number and decreased GSH level in CPAEC. The tested caspase inhibitors did not significantly change the number in PG-treated CPAEC. Each caspase inhibitor differently alters GSH levels in CPAEC. In conclusion, PG inhibited the growth of endothelial cells, especially CPAEC via caspase-independent apoptosis. PG-induced CPAEC death was accompanied by ROS increase and GSH depletion.

The attenuation of MG132, a proteasome inhibitor, induced A549 lung cancer cell death by p38 inhibitor in ROS-independent manner.

MG132, as a proteasome inhibitor, can induce apoptotic cell death through formation of reactive oxygen species (ROS). In this study, we investigated the effects of MAPK (MEK, JNK, and p38) inhibitors on MG132-treated A549 lung cancer cells in relation to cell growth, cell death, ROS, and glutathione (GSH) levels. Treatment with 10 microM MG132 inhibited the growth of A549 cells at 24 h. MG132 also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; deltapsi(m)). ROS were not increased in MG132-treated A549 cells. MG132 increased GSH-depleted cell numbers and decreased GSH levels. MEK and JNK inhibitors did not strongly affect cell growth, cell death, ROS, and GSH levels in MG132-treated A549 cells. In contrast, p38 inhibitor reduced cell growth inhibition, apoptosis, and MMP (deltapsi(m)) loss by MG132. However, p38 inhibitor did not change ROS level and GSH content. In conclusion, MG132 inhibited the growth of A549 cells via apoptosis without formation of ROS. Treatment with p38 inhibitor rescued some cells from MG132-induced apotposis, which was not affected by ROS and GSH level changes.

Gallic acid inhibits the growth of HeLa cervical cancer cells via apoptosis and/or necrosis.

Gallic acid (GA) is widely distributed in various plants and foods, and its various biological effects have been reported. Here, we evaluated the effects of GA on HeLa cells in relation to cell growth inhibition and death. HeLa cell growth was diminished with an IC(50) of approximately 80 microM GA at 24h whereas an IC(50) of GA in human umbilical vein endothelial cells (HUVEC) was approximately 400 microM. GA-induced apoptosis and/or necrosis in HeLa cells and HUVEC, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsi(m)). The percentages of MMP (DeltaPsi(m)) loss cells and death cells were lower in HUVEC than HeLa cells. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from GA-induced cell death. GA increased reactive oxygen species (ROS) level and GSH (glutathione) depleted cell number in HeLa cells. Caspase inhibitors reduced GSH depleted cell number but not ROS level in GA-treated HeLa cells. In conclusion, GA inhibited the growth of HeLa cells and HUVEC via apoptosis and/or necrosis. The susceptibility of HeLa cells to GA was higher than that of HUVEC. GA-induced HeLa cell death was accompanied by ROS increase and GSH depletion.

Propyl gallate inhibits the growth of HeLa cells via caspase-dependent apoptosis as well as a G1 phase arrest of the cell cycle.

Propyl gallate (PG) as a synthetic antioxidant exerts a variety of effects on tissue and cell functions. Here, we evaluated the effects of PG on the growth of HeLa cells in relation to apoptosis and the cell cycle. PG dose-dependently inhibited the growth of HeLa cells with an IC50 of approximately 800 microM at 24 h. DNA flow cytometric analysis indicated that PG significantly induced a G1 phase arrest of the cell cycle along with an increase in the cyclin-dependent kinase inhibitor (CDKI) p27. In addition, PG induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; DeltaPsim), activation of caspase-3 and caspase-8 and PARP cleavage. All the tested caspase inhibitors (pan-caspase, caspase-3, -8 or -9 inhibitor) significantly rescued HeLa cells from PG-induced cell death. However, none of the caspase inhibitors prevented the loss of MMP (DeltaPsim) induced by PG. In conclusion, PG inhibited the growth of HeLa cells via caspase-dependent apoptosis as well as a G1 phase arrest of the cell cycle.