Suvajit Sen

Role of cystathionine β-synthase in human breast Cancer

Cystathionine β-synthase (CBS) is an enzyme in the transulfuration pathway that can catalyze the condensation of homocysteine (Hcy) and cysteine (Cys) to hydrogen sulfide (H2S) and cystathionine (CTH). CBS-derived H2S is important in angiogenesis and drug resistance in colon and ovarian cancers, respectively. However, the mechanisms by which cancer cell-derived H2S is utilized by cancer cells as a protective agent against host-derived activated macrophages are not yet investigated. This study investigated the mechanistic role of CBS-derived H2S in the protection of human breast cancer (HBC) cells against activated macrophages. HBC patient-derived tissue arrays and immunoblot analysis of HBC cells exhibited significantly increased levels of CBS when compared with their normal counterparts. This was associated with increased levels of H2S and CTH. Silencing of CBS in HBC cells caused a significant decrease in the levels of H2S and CTH but did not affect the growth of these cells per se, in in vitro cultures. However CBS-silenced cells exhibited significantly reduced growth in the presence of activated macrophages and in xenograft models. This was associated with an increase in the steady state levels of reactive aldehyde-derived protein adducts. Exogenous addition of H2S countered the effects of CBS silencing in the presence of macrophages. Conversely overexpression of CBS in human breast epithelial (HBE) cells (which do not naturally express CBS) protected them from activated macrophages, which were otherwise susceptible to the latter.

Maintenance of higher H2O2 levels, and its mechanism of action to induce growth in breast cancer cells: Important roles of bioactive catalase and PP2A

We assessed the catalase bioactivity and hydrogen peroxide (H₂O₂) production rate in human breast cancer (HBC) cell lines and compared these with normal human breast epithelial (HBE) cells. We observed that the bioactivity of catalase was decreased in HBC cells when compared with HBE cells. This was also accompanied by an increase in H₂O₂ steady-state levels in HBC cells. Silencing the catalase gene led to a further increase in the steady-state level of H₂O₂ which was also accompanied by an increase in growth rate of HBC cells. Catalase activity was up regulated on treatment with superoxide (O₂⁻) scavengers such as pegylated SOD (PEG-SOD, indicating inhibition of catalase by the increased O₂⁻ produced by HBC cells. Transfection of either catalase or glutathione peroxidase to HBC cells decreased intracellular H₂O₂ levels and led to apoptosis of these cells. The H₂O₂ produced by HBC cells inhibited PP2A activity accompanied by increased phosphorylation of Akt and ERK1/2. The importance of catalase bioactivity in breast cancer was further confirmed as its bioactivity was also decreased in human breast cancer tissues when compared to normal breast tissues. We conclude that inhibition of catalase bioactivity by O₂⁻ leads to an increase in steady-state levels of H₂O₂ in HBC cells, which in turn inhibits PP2A activity, leading to phosphorylation of ERK 1/2 and Akt and resulting in HBC cell proliferation.

Mitochondrial-associated nitric oxide synthase activity inhibits cytochrome c oxidase: Implications for breast Cancer

Nitric oxide (NO) is produced and nitric oxide synthase (NOS) activity is expressed in many types of tumor cells, but their precise role in tumor proliferation has not been clearly elucidated. Recently, it has been observed that patients with triple-negative breast tumors expressing NOS have a significantly worse prognosis compared to those that do not express any NOS. We observed that NOS activity was associated with the mitochondria in two breast cancer cell lines, ZR-75-30 and BT-474, compared with another NO-producing benign breast epithelial cell line, MCF-12F, in which no significant mitochondrial-associated NOS activity was detected. The rate of proliferation of the malignant cells expressing mitochondrial-associated NOS was decreased in the presence of an inhibitor of NO synthesis, but it had no effect on the normal breast epithelial cells, MCF-12F, which also expressed NOS, but not associated with mitochondria. The basal rate of proliferation was not affected by ODQ, an inhibitor of soluble guanylate cyclase, indicating that the effects of the endogenous NO produced by the malignant cell lines on proliferation are cGMP independent. Our results indicate that mitochondrial-associated NOS activity exhibited by the cancer cell lines ZR-75-30 and BT-474 inhibited cytochrome c oxidase, resulting in increased production of hydrogen peroxide (H2O2), which inhibited protein phosphatase 2A activity. This resulted in the maintenance of Akt and ERK1/2 in a phosphorylated state, leading to cell proliferation.

Protein Phosphatase 2A Promotes Endothelial Survival via Stabilization of Translational Inhibitor 4E-BP1 Following Exposure to Tumor Necrosis Factor-α

Objective—Tumor necrosis factor-&agr; (TNF&agr;) may change from a stimulator of reversible activation of endothelial cells (ECs) to a killer when combined with cycloheximide (CHX). The means by which endothelial cells are destined to either the survival pathway or the apoptotic pathway are not fully understood. We investigated the role of p38 mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) activation and their regulation of 4E-BP1 stability in ECs to determine whether this pathway contributes to apoptosis induced by TNF&agr; and CHX. Methods and Results—Apoptosis was induced in human umbilical vein ECs (HUVECs) by treating them with a combination of TNF&agr; and CHX (TNF&agr;/CHX). Activation of p38 MAPK was increased in HUVECs undergoing apoptosis, which was associated with degradation of eukaryotic initiation factor 4A regulator 4E-BP1 in a p38 MAPK–dependent manner. CHX attenuated a TNF&agr;-stimulated increase in the expression and activity of PP2A. Silencing PP2A expression with small interfering RNA transfection mimicked CHX sensitization, increasing HUVEC apoptosis with TNF&agr; stimulation and suggesting a protective role for PP2A in the apoptotic process. Conclusion—Our data suggest that (1) TNF&agr; stimulates PP2A and HUVECs elude apoptosis by PP2A-dependent dephosphorylation of p38 MAPK, and (2) CHX-induced inhibition of PP2A leads to maintenance of p38 activity and degradation of 4E-BP1, resulting in enhanced TNF&agr;-induced apoptosis.

Gestational Diabetes, Preeclampsia and Cytokine Release: Similarities and Differences in Endothelial Cell Function

Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy affects the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. Similarly, some but not all studies have suggested that infection of the mother under certain circumstances can also lead to preeclampsia as women with either a bacterial or viral infection were at a higher risk of developing preeclampsia, compared to women without infection and infection also leads to a release in TNFα. Endothelial cells exposed to either high glucose or TNFα leads to an increase in the production of H2O2 and to a decrease in endothelial cell proliferation. The cellular and molecular mechanisms involved in this phenomenon are discussed.Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy has profound effects on the fetus and long term effects on the neonate. All three conditions affect the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. It has also been suggested although not universally accepted that under certain circumstances maternal infection may also predispose to pre-eclampsia. Pre-eclampsia is also associated with the release of TNFα and endothelial dysfunction. However, the cellular and molecular mechanism(s) leading to the endothelial dysfunction by either hyperglycemia or by the cytokine TNFα appear to be different. In this chapter, we explore some of the similarities and differences leading to endothelial dysfunction by both hyperglycemia and by the inflammatory cytokine TNFα and the cellular and molecular mechanism(s) involved.

γ-Aminobutyric Acid Is Synthesized and Released by the Endothelium Potential Implications

RATIONALE Gamma aminobutyric acid (GABA), a neurotransmitter of the central nervous system, is found in the systemic circulation of humans at a concentration between 0.5 and 3 μmol/L. However, the potential source of circulating GABA and its significance on the vascular system remains unknown. We hypothesized that endothelial cells (ECs) may synthesize and release GABA to modulate some functions in the EC and after its release into the circulation. OBJECTIVE To assess whether GABA is synthesized and released by the EC and its potential functions. METHODS AND RESULTS Utilizing the human umbilical vein ECs and aortic ECs, we demonstrated for the first time that ECs synthesize and release GABA from [1-(14)C]glutamate. Localization of GABA and the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase in EC were confirmed by immunostaining and immunoblot analysis, respectively. The presence of GABA was further confirmed by immunohistochemistry in the EC lining the human coronary vessel. EC-derived GABA regulated the key mechanisms of ATP synthesis, fatty acid, and pyruvate oxidation in EC. GABA protected EC by inhibiting the reactive oxygen species generation and prevented monocyte adhesion by attenuating vascular cell adhesion molecule -1 and monocyte chemoattractant protein-1 expressions. GABA had no relaxing effect on rat aortic rings. GABA exhibited a dose-dependent fall in blood pressure. However, the fall in BP was abolished after pretreatment with pentolinium. CONCLUSIONS Our findings indicate novel potential functions of endothelium-derived GABA.

Acid Gradient across Plasma Membrane Can Drive Phosphate Bond Synthesis in Cancer Cells: Acidic Tumor Milieu as a Potential Energy Source

Aggressive cancers exhibit an efficient conversion of high amounts of glucose to lactate accompanied by acid secretion, a phenomenon popularly known as the Warburg effect. The acidic microenvironment and the alkaline cytosol create a proton-gradient (acid gradient) across the plasma membrane that represents proton-motive energy. Increasing experimental data from physiological relevant models suggest that acid gradient stimulates tumor proliferation, and can also support its energy needs. However, direct biochemical evidence linking extracellular acid gradient to generation of intracellular ATP are missing. In this work, we demonstrate that cancer cells can synthesize significant amounts of phosphate-bonds from phosphate in response to acid gradient across plasma membrane. The noted phenomenon exists in absence of glycolysis and mitochondrial ATP synthesis, and is unique to cancer. Biochemical assays using viable cancer cells, and purified plasma membrane vesicles utilizing radioactive phosphate, confirmed phosphate-bond synthesis from free phosphate (Pi), and also localization of this activity to the plasma membrane. In addition to ATP, predominant formation of pyrophosphate (PPi) from Pi was also observed when plasma membrane vesicles from cancer cells were subjected to trans-membrane acid gradient. Cancer cytosols were found capable of converting PPi to ATP, and also stimulate ATP synthesis from Pi from the vesicles. Acid gradient created through glucose metabolism by cancer cells, as observed in tumors, also proved critical for phosphate-bond synthesis. In brief, these observations reveal a role of acidic tumor milieu as a potential energy source and may offer a novel therapeutic target.

Endothelial Cell Function in Utero-placental Circulation Physiology and Pathophysiology

Endothelial cells in the utero-placental circulation play an important physiological role in maintaining the fetoplacental vessels in a vasodilated state as these vessels are non-innervated. These endothelial cells produce both prostacyclin and nitric oxide which in addition to causing vasodilation also prevent platelet aggregation and adhesion of platelets to endothelial cells. Most investigators are of the opinion that energy metabolism of endothelial cells and ATP generation is mainly glycolytic. Glycolytic activity in endothelial cells is increased during proliferation to maintain ATP at normal levels by an increase in the expression of the glucose transporter. More recent studies have reported the existence of a functional F1F0 ATP synthase on the surface of HUVEC and it has been found to be enzymatically active in the synthesis of ATP. Additional studies utilizing very early passage HUVEC need to be carried out to ascertain the relative contribution of oxidative phosphorylation compared with the glycolytic pathway for ATP synthesis in normal pregnancy as well as in abnormal states like preeclampsia, diabetes, intrauterine injection as well as intrauterine growth restriction.

A light-activated NO donor attenuates anchorage independent growth of cancer cells: Important role of a cross talk between NO and other reactive oxygen species

It is established that high concentrations of nitric oxide(1) (NO), as released from activated macrophages, induce apoptosis in breast cancer cells. In this study, we assessed the potential of a light-activated NO donor [(Me2bpb)Ru(NO)(Resf)], a recently reported apoptototic agent, in suppressing the anchorage independent growth potentials of an aggressive human breast cancer cell line. Our results demonstrated the down regulation of anchorage independent growth by light activated NO treatment in the aggressive human breast cancer cell line MDA-MB-231 and afforded insight into the associated mechanism(s). The investigation revealed an up-regulation of the bioactivity of catalase with an accompanied reduction in the endogenous levels of H2O2, a direct substrate of catalase and a recently identified endogenous growth modulator in breast cancer cells. An earlier publication reported that endogenous superoxide (O2(-)) in human breast cancer cells constitutively inhibits catalase bioactivity (at the level of its protein), resulting in increased H2O2 levels. Interestingly in this study, O2(-) was also found to be down- regulated following NO treatment providing a basis for the observed increase in catalase bioactivity. Cells silenced for the catalase gene exhibited compromised reduction in anchorage independent growth upon light activated NO treatment. Collectively this study detailed a mechanistic cross talk between exogenous NO and endogenous ROS in attenuating anchorage independent growth.

Attenuation of Antioxidant Capacity in Human Breast Cancer Cells by Carbon Monoxide Through Inhibition of Cystathionine β-synthase Activity: Implications in Chemotherapeutic Drug Sensitivity

Drug resistance is a major impediment to effective treatment of breast cancer. Compared to normal cells, cancer cells have an increased antioxidant potential due to an increased ratio of reduced to oxidized glutathione (GSH/GSSG). This is known to confer therapeutic resistance. Here, we have identified a mechanism, unique to breast cancer cells, whereby cystathionine β-synthase (CBS) promotes elevated GSH/GSSG. Lentiviral silencing of CBS in human breast cancer cells attenuated GSH/GSSG, total GSH, nuclear factor erythroid 2-related factor 2 (Nrf2), and processes downstream of Nrf2 that promote GSH synthesis and regeneration of GSH from GSSG. Carbon monoxide (CO) reduced GSH/GSSG in three breast cancer cell lines by inhibiting CBS. Furthermore, CO sensitized breast cancer cells to doxorubicin. These results provide insight into mechanism(s) by which CBS increases the antioxidant potential and the ability for CO to inhibit CBS activity to alter redox homeostasis in breast cancer, increasing sensitivity to a chemotherapeutic.