Hydrogen Sulfide-releasing Aspirin Research Articles

MZe786, a hydrogen sulfide-releasing aspirin prevents preeclampsia in heme oxygenase-1 haplodeficient pregnancy under high soluble flt-1 environment

Preeclampsia affects one in twelve of the 130 million pregnancies a year. The lack of an effective therapeutic to prevent or treat it is responsible for an annual global cost burden of 100 billion US dollars. Preeclampsia also affects these women later in life as it is a recognised risk factor for cardiovascular disease, stroke and vascular dementia. Our laboratory demonstrated that preeclampsia is associated with high soluble fms-like tyrosine kinase 1 (sFlt-1) and low heme oxygenase-1 (HO1/Hmox1) expression. Here we sought to determine the therapeutic value of a novel H2S-releasing aspirin (MZe786) in HO-1 haploid deficient (Hmox1+/−) pregnant mice in a high sFlt-1 environment. Pregnant Hmox1+/− mice were injected with adenovirus encoding sFlt-1 or control virus at gestation day E11.5. Subsequently, Hmox1+/− dams were treated daily with a number of treatment regimens until E17.5, when maternal and fetal outcomes were assessed. Here we show that HO-1 compromised mice in a high sFlt-1 environment during pregnancy exhibit severe preeclampsia signs and a reduction in antioxidant genes. MZe786 ameliorates preeclampsia by reducing hypertension and renal damage possibly by stimulating antioxidant genes. MZe786 also improved fetal outcome in comparison with aspirin alone and appears to be a better therapeutic agent at preventing preeclampsia than aspirin alone.

Effect of hydrogen sulfide-releasing aspirin on esophageal and gastric mucosa compromised by stress injury

Effect of hydrogen sulfide-releasing aspirin on esophageal and gastric mucosa compromised by stress injury

Efficient cocktail chemotherapy by co-delivery of a hydrogen sulfide-releasing aspirin prodrug and paclitaxel via single nanoparticles

A cocktail composed of hydrogen sulfide-releasing aspirin (HS-ASP) and paclitaxel (PTX) in a single delivery system was formulated and provides a promising platform for the cocktail treatment of non-small cell lung cancer.

Hydrogen Sulfide Inhibits Human Platelet Aggregation In Vitro in Part by Interfering Gap Junction Channels: Effects of ACS14, a Hydrogen Sulfide-releasing Aspirin

Hydrogen sulfide (H2S), as a newly identified gaseous mediator, has been widely investigated in various systems. However, the effect of H2S on cardiovascular system haemostasis, including platelet aggregation and the precise mechanisms remain unclear. Therefore, the present study was aimed to examine the inhibitory effect of H2S on human platelet aggregation in vitro and its relevance to gap junction channels. The antiaggregatory property of H2S-releasing aspirin derivative (ACS14) was compared with its mother compound, aspirin. In comparison to an equimolar dose of aspirin, ACS14 not only exerted a more potent inhibitory effect on platelet aggregation induced by ADP or thrombin, but also significantly inhibited αIIbβ3 integrin activation and P-selectin expression. Similarly, NaHS (100μM), a conventional H2S donor significantly inhibited platelet aggregation as well as αIIbβ3 integrin activation and P-selectin expression induced by ADP or thrombin. Furthermore, pretreatment with rotigaptide, a gap junction modifier abolished the inhibitory properties of ACS14 or NaHS on platelet aggregation, suggesting that suppression of platelet aggregation by H2S is, at least in part, gap junction channel-dependent. H2S may inhibit human platelet aggregation at least in part by depressing gap junction intercellular communication and H2S released from ACS14 may contribute to its additional anti-platelet effect in vitro in comparison to aspirin.

Hydrogen sulfide-releasing aspirin inhibits the growth of leukemic Jurkat cells and modulates β-catenin expression

Hydrogen sulfide-releasing aspirin (HS-ASA) is a novel compound with potential against cancer. It inhibited the growth of Jurkat T-leukemia cells with an IC₅₀ of 1.9 ± 0.2 μM whereas that of ASA was >5000 μM. It dose-dependently inhibited proliferation and induced apoptosis in these cells, causing a G₀/G₁ cell cycle arrest. HS-ASA down-regulated β-catenin protein levels and reduced mRNA and protein expression of β-catenin/TCF downstream target genes cyclinD1 and c-myc. Aspirin up to 5 mM had no effect on β-catenin expression. HS-ASA also increased caspase-3 protein levels and dose-dependently increased its activity. These effects were substantially blocked by z-VAD-fmk, a pan-caspase inhibitor.

Abstract 3891: NOSH-aspirin, a novel nitric oxide- and hydrogen sulfide-releasing hybrid, is a potent inhibitor of colon cancer cell growth

Abstract Introduction: Non-steroidal anti-inflammatory drugs (NSAIDs) are recognized as the prototypical chemopreventive agents. However, their wide use is precluded due to significant toxicity. The search for better NSAIDs has led to generation of nitric oxide-releasing NSAIDs (NO-NSAIDs) and more recently, to a new class of hydrogen sulfide-releasing agents (HS-NSAIDs). Both of these have shown to be more potent and less toxic than their parent NSAID. However, the draw back to these compounds is their IC50 for cell growth inhibition. It is too high to make them attractive for development as pharmaceuticals. Therefore, we postulated that a new hybrid that incorporated the active parts of each compound may be more potent and effective than either one. Our hypothesis has proved to be correct. Here we describe the effects of a nitric oxide- and hydrogen sulfide-releasing aspirin (NOSH-aspirin) on the growth properties of several human colon cancer cell lines, on induction of reactive oxygen species (ROS), and on NF-κB whose induction is strongly implicated in some cancers. Methods: NOSH-aspirin was synthesized and purified at our lab with 1H-NMR verification. Colon cancer cell lines: HT-29, HCT 15, and SW480; Cell growth: MTT; Cell cycle phase distribution: Flow cytometry; Apoptosis: subdiploid (sub-G0/G1) peak in DNA content histograms; Proliferation: PCNA; NF-kB: enzyme-linked immunosorbent assay (ELISA); Reactive oxygen species (ROS): 2′,7′-dichlorodihydrofluorescein (H2DCFDA, probe for peroxides) or dihydroethidium (DHE, probe for superoxide anion) measured fluorescence by flow cytometry. Results: NOSH-aspirin inhibited the growth of HT-29, HCT 15, and SW480 cells with IC50 values of 48 ± 3, 50 ± 5, and 60 ± 4 nM at 24 hr, respectively. The corresponding IC50 for aspirin in all 3 cell lines was >5000 µM. This represents and enhanced potency of >80,000-fold in the 3 cell lines at 24 h. NOSH-aspirin treatment of HT-29 cells at 0.5xIC50, 1xIC50, and 2xIC50 for 24 hrs induced apoptosis (20 ± 1%, 52 ± 3%, 75 ± 3%), inhibited proliferation (PCNA, 70 ± 3%, 45 ± 4%, 22 ± 3%), and caused a G0/G1 cell cycle block. Activation of NF-κB was inhibited as demonstrated by ELISA, at 0.5xIC50, 1xIC50, and 2xIC50 the reduction was 11 ± 1%, 33 ± 2%, and 46 ± 3% respectively. NOSH-aspirin caused induction of oxidative stress as evidenced by increases in both H2DCFDA and DHE-derived fluorescence representing increases in intracellular levels of H2O2 and superoxide anion respectively Conclusions: NOSH-aspirin by inducing ROS is a potent inhibitor of colon cancer cell growth and it inhibits NF-κB activation. These data suggest that NOSH-aspirin may be useful as a chemopreventive agent against colon cancer and merits further evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3891. doi:1538-7445.AM2012-3891

Hydrogen sulfide-releasing aspirin modulates xenobiotic metabolizing enzymes in vitro and in vivo

The balance between phase-I carcinogen-activating and phase-II detoxifying xenobiotic metabolizing enzymes is critical to determining an individual's risk for cancer. We evaluated the effect of Hydrogen sulfide-releasing aspirin (HS-ASA) on xenobiotic metabolizing enzymes in HT-29 human colon and Hepa 1c1c7 mouse liver adenocarcinoma cells and in Wistar rats. HS-ASA inhibited the growth of HT-29 and Hepa 1c1c7 cells, with an IC50 of 3.2±0.3μM and 4.2±0.4μM, respectively. The IC50 for ASA in both cell lines was greater than 5000μM at 24h. In these cell lines, HS-ASA caused a dose-dependent increase in activity and expression of the phase-II enzymes glutathione S-transferase (GST) and NAD(P)H:quinoneoxireductase (NQO1). It also caused an increase in UDP-glucuronosyltransferase (UGT) expression. The levels of CYP 1A1 a phase-I enzyme was increased by HS-ASA in both cell lines. Pretreatment of cells with NaF, an esterase inhibitor, abrogated the HS-ASA-mediated increases in NQO1 enzyme activity. HS-ASA increased the protein levels of the transcription factor Nrf2, which is a regulator of the phase-II enzymes. In vivo, HS-ASA at 100mg/kg/day had no effect on rat's weights; it induced a 3.4-fold and 1.4-fold increase in hepatic GST and NQO1 enzyme activities, respectively. GST and NQO1 protein levels were also increased. In contrast to that in cultured cells, CYP 1A1 protein levels were not altered in vivo. Therefore, HS-ASA induces phase-II enzymes, at least in part, through the action of H2S and by modulating Nrf2; these effects may be part of its mechanism of action against carcinogenesis.

Hydrogen sulfide-releasing aspirin suppresses NF-κB signaling in estrogen receptor negative breast cancer cells in vitro and in vivo

Hormone-dependent estrogen receptor positive (ER+) breast cancers generally respond well to anti-estrogen therapy. Unfortunately, hormone-independent estrogen receptor negative (ER−) breast cancers are aggressive, respond poorly to current treatments and have a poor prognosis. New approaches and targets are needed for the prevention and treatment of ER− breast cancer. The NF-κB signaling pathway is strongly implicated in ER− tumor genesis, constituting a possible target for treatment. Hydrogen sulfide-releasing aspirin (HS-ASA), a novel and safer derivative of aspirin, has shown promise as an anti-cancer agent. We examined the growth inhibitory effect of HS-ASA via alterations in cell proliferation, cell cycle phase transitions, and apoptosis, using MDA-MB-231 cells as a model of triple negative breast cancer. Tumor xenografts in mice, representing human ER− breast cancer, were evaluated for reduction in tumor size, followed by immunohistochemical analysis for proliferation, apoptosis and expression of NF-κB. HS-ASA suppressed the growth of MDA-MB-231 cells by induction of G0/G1 arrest and apoptosis, down-regulation of NF-κB, reduction of thioredoxin reductase activity, and increased levels reactive oxygen species. Tumor xenografts in mice, were significantly reduced in volume and mass by HS-ASA treatment. The decrease in tumor mass was associated with inhibition of cell proliferation, induction of apoptosis and decrease in NF-κB levels in vivo. HS-ASA has anti-cancer potential against ER− breast cancer and merits further study.

Abstract 807: Hydrogen sulfide-releasing aspirin modulates xenobiotic metabolizing enzymes in vitro and in vivo

Abstract Introduction: Xenobiotic metabolizing enzymes are classified as phase-I (oxidation/reduction) and phase-II (conjugation) enzymes; the balance between the phase-I carcinogen-activating enzymes and the phase-II detoxifying enzymes may be important in determining an individual's risk for cancer. Hydrogen sulfide-releasing aspirin (HS-ASA) is a new compound with significant anti-inflammatory properties. It consists of ASA covalently attached to 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione, which releases hydrogen sulfide. Our initial studies indicate that HS-ASA has chemopreventive properties. In the present study, we evaluated the effect of HS-ASA on xenobiotic metabolizing enzymes both in vitro and vivo. Methods: HS-ASA was synthesized and purified by us with 1H-NMR verification. Cell lines: HT-29 human colon adenocarcinoma and Hepa 1c1c7 mouse liver adenocarcinoma. Cell growth inhibition: MTT. Levels of cytochrome P450 1A1 (CYP 1A1) a phase-1 enzyme and NAD(P)H:quinone oxireductase (NQO), glutathione S-transferase (GST) and UDP-glucuronyltransferase (UGT), phase-II enzymes by immunoblots. Enzyme activity: GST and thioredoxin reductase (Trx). In vivo: Male Wistar rats (N = 4/group) were treated via gavage either with vehicle or with HS-ASA 100 mg/kg/day. Weight of animals in each group was recorded every 3 days. After 21 days, animals were killed and liver microsomes and cytosols were prepared for phase-I and phase-II enzyme evaluation. Results: HS-ASA inhibited the growth of HT-29 and Hepa 1c1c7 cells, with an IC50 of 2.5 ± 0.3 µM and 2.9 ± 0.4 µM, respectively. The IC50 for ASA in both cell lines was >5,000 µM at 24h. The ratio ASA/HS-ASA is >2000 in the HT-29 cells and >1700 in Hepa 1c1c7 cells, suggesting that HS-ASA is at least 1700-2000-fold more potent than traditional ASA. In HT-29 cells, at 0.5xIC50, 1xIC50, and 2xIC50, HS-ASA increased GST activity by 2.5-, 7.2-, and 11-fold; and it inhibited Txr by 36 ± 3%, 67 ± 2% and 79 ± 2% respectively. In HT-29 cells, levels of CYP 1A1 were not significantly affected whereas levels of GST and UGT were increased dose-dependently by HS-ASA. In Hepa 1c1c7 cells, levels of NQO were also significantly increased. In vivo, HS-ASA: 1. had no effect on rat's weight; 2. increased hepatic GST activity by 3.4-fold and NQO by 1.4-fold; 3. immunoblots showed significant increases in GST and NQO protein levels but no changes in CYP 1A1 protein levels. Conclusions: HS-ASA co-induces multiple phase-II metabolizing enzymes and thus may be regarded as a monofunctional inducer. This represents one mechanism by which HS-ASA exhibits chemopreventive properties. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 807. doi:10.1158/1538-7445.AM2011-807

Abstract 1349: Hydrogen sulfide-releasing aspirin suppresses NF-κB signaling in estrogen receptor negative breast cancer cells in vitro and in vivo

Abstract Introduction: Estrogen receptor negative [ER(−)] breast cancer is very aggressive, responds poorly to current treatments and has a poor prognosis. The NF-κB signaling pathway is strongly implicated in ER(−) tumorigenesis. Epidemiological studies show a chemopreventive effect for aspirin (ASA) against ER(+) but not for ER(−) breast cancers. Hydrogen sulfide-releasing aspirin (HS-ASA) is a safer ASA consisting of an ASA molecule to which an H2S-releasing moiety (5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH) has been covalently attached. We have observed that HS-ASA is very potent in inhibiting the growth of ER(−) breast cancer cells an effect which is modulated by NF-κB. Methods: HS-ASA was synthesized and purified at our lab with 1H-NMR verification. Cell growth: MTT; Cell cycle phase distribution: Flow cytometry; Apoptosis: subdiploid (sub-G0/G1) peak in DNA content histograms; Proliferation: PCNA; NF-κB: enzyme-linked immunosorbent assay (ELISA). Xenografts: Male athymic SKID mice were implanted s.c. in the right flank with MDA-MB-231 (3 × 106) cells suspended in 50% Matrigel, after 5 days the animals were randomly divided into 2 groups (N = 6/group) and gavaged daily with HS-ASA (100 mg/kg body weight) or vehicle. Tumor volume and animal weight were recorded every 2 days. After 4 weeks of treatment, mice were sacrificed, tumors excised, weighed, and fixed in 10% buffered formalin for immunohistochemical studies. Results: HS-ASA inhibited the growth of MDA-MB-231 cells, an ER(−) human breast cancer cell line; at 24h the IC50 being 3.6 ± 0.5 µM whereas for ASA the IC50 was >5,000 µM. The ratio ASA/HS-ASA is >1000, meaning that HS-ASA is at least 1000 times more potent than traditional ASA. In cultured ER(−) breast cancer cells, HS-ASA treatment for 24 hrs showed the following: 1. There was a combined inhibition of cell proliferation, induction of apoptosis and a cell cycle block at G0/G1; PCNA expression was reduced dose dependently, at 0.5xIC50, 1xIC50, and 2xIC50 the reduction was 79 ± 2 %, 47 ± 3 % and 36 ± 2 % respectively; % of apoptotic cells at 0.5xIC50, 1xIC50, and 2xIC50 were 15 ± 2%, 43 ± 3%, and 64 ± 5% respectively; 2. Activation of NF-κB was inhibited as demonstrated by ELISA, at 0.5xIC50, 1xIC50, and 2xIC50 the reduction was 10 ± 2%, 31 ± 3%, and 50 ± 3% respectively. Xenografts: 1. HS-ASA had no effect on the weight of the mice; 2. HS-ASA reduced tumor mass by 55 ± 7% and tumor volume as a function of time; 3. HS-ASA inhibited growth of ER(−) breast cancer cells as a result of reduced proliferation (decreased PCNA expression), and induction apoptosis (increased number of TUNEL positive cells); 4. NF-κB, activated in control xenografts was significantly inhibited by HS-ASA. Conclusions: HS-ASA inhibits NF-κB which is central in inflammatory cascades. This suggests that HS-ASA may have a role in chemoprevention, in particular in ER(−) breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1349. doi:10.1158/1538-7445.AM2011-1349

Abstract 4608: Hydrogen sulfide-releasing aspirin inhibits the growth of leukemic Jurkat cells and modulates β-catenin expression

Abstract Introduction: Acute leukemia is the primary cause of cancer-related mortality in children. ß -catenin, a regulator of cell-cell adhesion and transcription, is expressed in T-acute lymphoblastic leukemia cells, tumor lines of hematopoietic origin and primary leukemia cells but not in normal peripheral blood T cells. ß -catenin may promote leukemia cell proliferation, adhesion, and survival. Hydrogen sulfide-releasing aspirin (HS-ASA) is a novel compound with significant potential for the control of cancer. It consists of a traditional ASA molecule covalently bound to an H2S-releasing moiety (5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione). Here we examined the effect of HS-ASA on the growth of Jurkat T-acute lymphoblastic leukemia cells and on the fate of ß -catenin. Methods: HS-ASA was synthesized and purified at our lab with 1H-NMR verification. Cell line: Jurkat T-leukemia. Quantification of cell growth: MTT assay. Cell kinetics: cell cycle phase distribution by flow cytometry; apoptosis by subdiploid (sub-G0/G1) peak in DNA content histograms; and proliferation by PCNA. ß-Catenin and caspase-3 expression was assayed by immunoblotting in cells treated with HS-ASA at 24 hr. Caspase-3 enzyme activity, and H2S levels was also measured. Results: 1) The IC50 for HS-ASA was 1.5 ± 0.2 µM, whereas that of ASA was >5000 µM strongly suggesting that HS-ASA is at least 3000-fold more potent than traditional ASA. 2) At 0.5xIC50, 1xIC50, and 2xIC50 the following observations were made: Proliferation was inhibited by 88 ± 2%, 63 ± 2%, 42 ± 3%; apoptosis was increased by 15 ± 2%, 39 ± 3%, 54 ± 4%; and cells in the G0/G1 phase increased dose-dependently whereas those in the S and G2/M phases decreased, suggesting a G0/G1 block. 3) HS-ASA dose-dependently reduced the levels of ß-catenin, whereas ASA up to 5 mM had no effect on ß-catenin expression. HS-ASA also increased caspase-3 protein levels and increased its activity dose-dependently, at 0 (no treatment), 0.5xIC50, 1xIC50, and 2xIC50 the values were 3.9 ± 0.3, 12.3 ± 0.5, 23.3 ± 0.7, and 42.6 ± 1.2 pmol/min/mg protein. H2S levels also increased as a function of HS-ASA concentration. Conclusion: HS-ASA strongly inhibits the growth of Jurkat T cells and causes inhibition of ß-catenin expression. These studies suggest one mechanism by which HS-ASA inhibits the growth of Jurkat T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4608. doi:10.1158/1538-7445.AM2011-4608

Activity of a new hydrogen sulfide-releasing aspirin (ACS14) on pathological cardiovascular alterations induced by glutathione depletion in rats

We investigated the effects of the hydrogen sulfide (H 2S)-releasing derivatives of aspirin (ACS14) and salicylic acid (ACS21) in a rat model of metabolic syndrome induced by glutathione (GSH) depletion, causing hypertension and other pathological cardiovascular alterations. GSH depletion was induced in normal rats by the GSH-synthase inhibitor buthionine sulfoximine (BSO, 30 mmol/L day for seven days in the drinking water). Systolic blood pressure and heart rate were measured daily by the tail-cuff method, and plasma thromboxane B 2, 6-keto-prostaglandin F 2α, 8-isoprostane, GSH, insulin and glucose were determined at the end of the seven-day BSO schedule. In addition, ischemia/reperfusion-induced myocardial dysfunction and endothelial dysfunction were assayed on isolated heart and aortic rings, respectively. Unlike aspirin and salicylic acid, ACS14 and ACS21 reduced BSO-induced hypertension, also lowering plasma levels of thromboxane B 2, 8-isoprostane and insulin, while GSH remained in the control range. Neither ACS14 nor ACS21 caused gastric lesions. Both restored the endothelial dysfunction observed in aortic rings from BSO-treated rats, and in ischemia/reperfusion experiments they lowered left ventricular end-diastolic pressure, consequently improving the developed pressure and the maximum rise and fall of left ventricular pressure. Together with this improvement of heart mechanics there were reductions in the activity of creatine kinase and lactate dehydrogenase in the cardiac perfusate. This implies that H 2S released by both ACS14 and ACS21 was involved in protecting the heart from ischemia/reperfusion, and significantly limited vascular endothelial dysfunction in aortic tissue and the related hypertension.