Khosrow Kashfi

Biology and therapeutic potential of hydrogen sulfide and hydrogen sulfide-releasing chimeras

Hydrogen sulfide, H2S, is a colorless gas with a strong odor that until recently was only considered to be a toxic environmental pollutant with little or no physiological significance. However, the past few years have demonstrated its role in many biological systems and it is becoming increasingly clear that H2S is likely to join nitric oxide (NO) and carbon monoxide (CO) as a major player in mammalian biology. In this review, we have provided an overview of the chemistry and biology of H2S and have summarized the chemistry and biological activity of some natural and synthetic H2S-donating compounds. The naturally occurring compounds discussed include, garlic, sulforaphane, erucin, and iberin. The synthetic H2S donors reviewed include, GYY4137; cysteine analogs; S-propyl cysteine, S-allyl cysteine, S-propargyl cysteine, and N-acetyl cysteine. Dithiolethione and its NSAID and other chimeras such as, L-DOPA, sildenafil, aspirin, diclofenac, naproxen, ibuprofen, indomethacin, and mesalamine have also been reviewed in detail. The newly reported NOSH-aspirin that releases both NO and H2S has also been discussed.

Nitric Oxide-Donating Nonsteroidal Anti-Inflammatory Drugs Inhibit the Growth of Various Cultured Human Cancer Cells: Evidence of a Tissue Type-Independent Effect

The novel nitric oxide (NO)-donating nonsteroidal anti-inflammatory drugs (NO-NSAIDs), which are safer than their NSAID counterparts, inhibit the growth of colon cancer cells with far greater potency than traditional NSAIDs. We examined whether NO-NSAIDs inhibit the growth of cancer cells arising from other human tissues. Human pancreatic, colon, prostate, lung, and tongue cancer cell lines were treated with NO-aspirin, -sulindac, -ibuprofen, and -indomethacin or their traditional counterparts. We determined IC50 values, cell proliferation, apoptosis, cell cycle, cyclooxygenase (COX) protein levels, and morphological changes (light and electron microscopy). All NO-NSAIDs inhibited the growth of all cancer cell lines studied. The potency of NO-NSAIDs was 11- to 6000-fold greater than that of their counterparts (except for the effect of sulindac on lung cancer cells). NO-aspirin was consistently the most potent NO-NSAID in all cell lines tested (except for the lung cancer cell line), sometimes in excess of 100-fold over the other three NO-NSAIDs. NO-NSAIDs inhibited cell proliferation, induced apoptosis, and altered cell cycle phase distribution (G2/M to G0/G1 block). All altered cellular morphology, whereas NO-aspirin induced nuclear disintegration (“atypical” cells) established by electron microscopy. NO-aspirin showed similar effects on two pancreatic cancer cell lines, BxPC-3 (expresses COX) and MIA PaCa-2 (no COX expression), suggesting a COX-independent effect. NO-NSAIDs showed a tissue-type-independent effect. Their pleiotropic effects involve cell renewal, cell death, and cell cycle phase transitions. These results raise the possibility that NO-NSAIDs possess chemopreventive and/or chemotherapeutic activity against a wide variety of human cancers.

The effect of leukotrienes B and selected HETEs on the proliferation of colon cancer cells

Eicosanoids have been implicated in colon carcinogenesis, but very little is known on the potential role of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) in this process; such compounds are produced by colonocytes and tumor infiltrating leukocytes. We studied the effect of LTB4, LTB4 methyl ester, LTB5, 12(R)-HETE, 12(S)-HETE and 15(S)-HETE (10(-10), 10(-8), 10(-6) M) on the proliferation rate, the cell cycle distribution, and the rate of apoptosis in HT-29 and HCT-15 human colon carcinoma cells. Our data show that LTB4, a lipoxygenase product, increased the proliferation rate of both cell lines in a time- and concentration-dependent manner. In HT-29 cells the concentration-response curve was bell-shaped (maximal effect at 10(-8) M). The proliferative effects of LTB4 in HT-29 cells were inhibited by SC-41930, a competitive antagonist of LTB4, suggesting the existence of an LTB4 receptor in epithelial cells. The methyl ester of LTB4 stimulated the proliferation of these cells, but LTB5, an isomer of LTB4 derived from eicosapentaenoic acid, did not. Of the HETEs, only 12(R)-HETE, a P-450 product, stimulated the proliferation of both cell lines; the other HETEs, all lipoxygenase products, failed to affect the proliferation of these cells. None of these eicosanoids had any effect on cell cycle distribution or apoptosis in either cell line. Taken together with our previous data showing that PGs stimulate colon cancer cell proliferation (Qiao et al. (1995) Biochim. Biophys. Acta 1258, 215-223), these findings indicate that arachidonic acid products synthesized via at least three different pathways (cyclooxygenase, lipoxygenase, P-450) may not be able to modulate the growth of colon cancer, and suggest a potential role in human colon carcinogenesis for LTB4 and 12(R)-HETE.

Nitric oxide-donating aspirin inhibits β-catenin/T cell factor (TCF) signaling in SW480 colon cancer cells by disrupting the nuclear β-catenin–TCF association

Dysregulation of the Wnt pathway and altered β-catenin expression are central early events in colorectal carcinogenesis. We studied the ortho, meta, and para (o-, m-, and p-) positional isomers of NO-donating aspirin (NO-ASA), a chemopreventive agent against colon cancer, for their effect on β-catenin/T cell factor (TCF) signaling. In human SW480 colon carcinoma cells, cell-growth inhibition by NO-ASA [IC50 values for p-, o-, and mwere 48.1 ± 4.3 (mean ± SEM), 60.4 ± 2.1, and 900 ± 50 μM, respectively] was accompanied by significant inhibition of β-catenin signaling. We determined β-catenin-dependent TCF-4 transcriptional activity by measuring the activity of the luciferase gene placed under the control of TCF-4 regulatory sequences. The IC50 values for β-catenin/TCF-4-signaling inhibition by NO-ASA were: o-, 2.6 ± 0.4; m-, 15 ± 5; p-, 1.1 ± 0.1 μM; and for ASA, >5,000 μM. Total or nuclear levels of β-catenin and its distribution in the cell were not altered by NO-ASA, as judged by protein expression levels and semiquantitative immunofluorescence analysis. NO-ASA disrupted the association of β-catenin and TCF-4 in the nucleus, whereas ASA did not affect it. NO-ASA reduced the expression of cyclin D1, a downstream target gene that plays an important role in colon carcinogenesis. In contrast, a structural analog of NO-ASA lacking the —NO2 moiety did not affect TCF-4 transcriptional activity. Thus, NO-ASA inhibits β-catenin-mediated TCF activity by preventing the formation of the β-catenin/TCF-4 complex. This effect, occurring at NO-ASA concentrations far below those required to inhibit cell growth, may be a critical early event in the chemopreventive activity of NO-ASA against colon cancer.

NOSH-aspirin (NBS-1120), a novel nitric oxide- and hydrogen sulfide-releasing hybrid is a potent inhibitor of colon cancer cell growth in vitro and in a xenograft mouse model.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are prototypical anti-cancer agents. However, their long-term use is associated with adverse gastrointestinal effects. Recognition that endogenous gaseous mediators, nitric oxide (NO) and hydrogen sulfide (H(2)S) can increase mucosal defense mechanisms has led to the development of NO- and H(2)S-releasing NSAIDs with increased safety profiles. Here we report on a new hybrid, NOSH-aspirin, which is an NO- and H(2)S-releasing agent. NOSH-aspirin inhibited HT-29 colon cancer growth with IC(50)s of 45.5 ± 2.5, 19.7 ± 3.3, and 7.7 ± 2.2 nM at 24, 48, and 72 h, respectively. This is the first NSAID based agent with such high degree of potency. NOSH-aspirin inhibited cell proliferation, induced apoptosis, and caused G(0)/G(1) cell cycle block. Reconstitution and structure-activity studies representing a fairly close approximation to the intact molecule showed that NOSH-aspirin was 9000-fold more potent than the sum of its parts towards growth inhibition. NOSH-aspirin inhibited ovine COX-1 more than ovine COX-2. NOSH-ASA treatment of mice bearing a human colon cancer xenograft caused a reduction in volume of 85%. Taken together, these results demonstrate that NOSH-aspirin has strong anti-cancer potential and merits further evaluation.

Hydrogen sulfide-releasing NSAIDs inhibit the growth of human cancer cells: a general property and evidence of a tissue type-independent effect.

Hydrogen sulfide-releasing non-steroidal anti-inflammatory drugs (HS-NSAIDs) are an emerging novel class of compounds with significant anti-inflammatory properties. They consist of a traditional NSAID to which an H(2)S-releasing moiety is covalently attached. We examined the effects of four different HS-NSAIDs on the growth properties of eleven different human cancer cell lines of six different tissue origins. Human colon, breast, pancreatic, prostate, lung, and leukemia cancer cell lines were treated with HS-aspirin, -sulindac, -iburofen, -naproxen, and their traditional counterparts. HS-NSAIDs inhibited the growth of all cancer cell lines studied, with potencies of 28- to >3000-fold greater than that of their traditional counterparts. HS-aspirin (HS-ASA) was consistently the most potent. HS-NSAIDs inhibited cell proliferation, induced apoptosis, and caused G(0)/G(1) cell cycle block. Metabolism of HS-ASA by colon cells showed that the acetyl group of ASA was hydrolyzed rapidly, followed by hydrolysis of the ester bond linking the salicylate anion to the H(2)S releasing moiety, producing salicylic acid and ADT-OH from which H(2)S is released. In reconstitution studies, ASA and ADT-OH were individually less active than the intact HS-ASA towards cell growth inhibition. Additionally, the combination of these two components representing a fairly close approximation to the intact HS-ASA, was 95-fold less active than the intact HS-ASA for growth inhibition. Taken together, these results demonstrate that HS-NSAIDs have potential anti-growth activity against a wide variety of human cancer cells.

The dual role of iNOS in cancer

Nitric oxide (NO) is one of the 10 smallest molecules found in nature. It is a simple gaseous free radical whose predominant functions is that of a messenger through cGMP. In mammals, NO is synthesized by the enzyme nitric oxide synthase (NOS) of which there are three isoforms. Neuronal (nNOS, NOS1) and endothelial (eNOS, NOS3) are constitutive calcium-dependent forms of the enzyme that regulate neural and vascular function respectively. The third isoform (iNOS, NOS2), is calcium-independent and is inducible. In many tumors, iNOS expression is high, however, the role of iNOS during tumor development is very complex and quite perplexing, with both promoting and inhibiting actions having been described. This review will aim to summarize the dual actions of iNOS-derived NO showing that the microenvironment of the tumor is a contributing factor to these observations and ultimately to cellular outcomes.

Nitric Oxide–Donating Aspirin Prevents Pancreatic Cancer in a Hamster Tumor Model

To evaluate the chemopreventive effect of nitric oxide-donating aspirin (NO-ASA), an ASA bearing a NO-releasing moiety, against pancreatic cancer, we studied six groups of female Syrian golden hamsters: groups 1 to 3 (n = 12 each) were given saline and groups 4 to 6 (n = 17) the carcinogen N-nitrosobis(2-oxopropyl)amine (BOP) s.c. in five weekly injections (the first, 70 mg/kg, and the remaining, 20 mg/kg each). Control and BOP-treated hamsters were fed a NO-ASA 3,000 ppm or conventional ASA 3,000 ppm or control diet for 19 weeks. Groups 1 to 3 had no tumors. Compared with the BOP/vehicle group, NO-ASA reduced the incidence (88.9%, P < 0.003) and multiplicity (94%, P < 0.05) of pancreatic cancer; ASA had no statistically significant effect. NO-ASA arrested the transition from PanIN2 to PanIN3 and carcinoma. The proliferation (proliferating cell nuclear antigen) / apoptosis (terminal deoxyribonucleotide transferase-mediated nick-end labeling) ratio of ductal cells increased with the histologic severity of the ductal lesion; NO-ASA suppressed it significantly during all stages except PanIN1A. p21(WAF1/CIP1), undetectable in normal cells, was progressively induced in neoplastic cells and suppressed by NO-ASA up to PanIN3. Nuclear factor-kappaB activation, absent in normal tissue, increased progressively (17-fold in cancer); NO-ASA suppressed it throughout and significantly in PanIN1B and PanIN2. Cyclooxygenase-2 expression, absent during early stages, was induced 6-fold in carcinoma and suppressed by NO-ASA in PanIN3 and carcinoma. Conventional ASA had no effect on these molecular markers. Thus, NO-ASA profoundly prevented pancreatic cancer and modulated multiple molecular targets in this model system; conventional ASA had no such effects. NO-ASA merits further evaluation as a chemopreventive agent against pancreatic cancer.

Inhibition of Bacterial Superantigens by Peptides and Antibodies

ABSTRACT The pyrogenic exotoxins of group A streptococci and staphylococcal enterotoxins are a family of structurally related superantigens with similar biological activity. Two distinct areas have been identified which have a highly conserved amino acid homology in all of the toxin families. A number of peptides were constructed from these regions, some of which were concatenated and polymerized to enhance their immunogenicity in animals. Antibodies prepared against these polymerized peptides were used to serologically identify the majority of the superantigen toxins, block the biological activities of the superantigens, and protect an experimental animal model against shock. In addition certain peptides were able per se to block up to 90% of the proliferative responses induced by the toxins. The peptide also proved protective in a septic shock model in mice. Binding experiments indicate that the peptide binds tightly to the major histocompatibility complex class II molecule, thus preventing binding and hence activation of the superantigen. The selective and rapid binding of the peptide to the major histocompatibility complex class II molecule may lead to a novel therapeutic modality in treatment of superantigen-mediated diseases.

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 G(0)/G(1) 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.