Pioglitazone treatment increases the cellular acid-labile and protein-bound sulfane sulfur fractions.

BACKGROUND: Iron-sulfur clusters play a central role in cellular function and are regulated by the ATM protein. Iron-sulfur clusters are part of the cellular sulfide pool, which functions to maintain cardiovascular health, and consists of free hydrogen sulfide, iron-sulfur clusters, protein bound sulfides, which constitute the total cellular sulfide fraction. ATM protein signaling and the drug pioglitazone share some cellular effects, which led us to examine the effects of this drug on cellular iron-sulfur cluster formation. Additionally, as ATM functions in the cardiovasculature and its signaling may be diminished in cardiovascular disease, we examined pioglitazone in the same cell type, with and without ATM protein expression. METHODS: We examined the effects of pioglitazone treatment on the total cellular sulfide profile, the glutathione redox state, cystathionine gamma-lyase enzymatic activity, and on double-stranded DNA break formation in cells with and without ATM protein expression. RESULTS: Pioglitazone increased the acid-labile (iron-sulfur cluster) and bound sulfur cellular fractions and reduced cystathionine gamma-lyase enzymatic activity in cells with and without ATM protein expression. Interestingly, pioglitazone also increased reduced glutathione and lowered DNA damage in cells without ATM protein expression, but not in ATM wild-type cells. These results are interesting as the acid-labile (iron-sulfur cluster), bound sulfur cellular fractions, and reduced glutathione are low in cardiovascular disease. CONCLUSION: Here we found that pioglitazone increased the acid-labile (iron-sulfur cluster) and bound sulfur cellular fractions, impinges on hydrogen sulfide synthesis, and exerts beneficial effect on cells with deficient ATM protein signaling. Thus, we show a novel pharmacologic action for pioglitazone.

Metastatic TFE3-overexpressing renal clear cell carcinoma with dense granules: a histological, immunohistochemical, and ultrastructural study.

This is a case report of a 46-year-old white male who presented with dyspnea. Thoracic and abdominal examinations showed a heterogeneously enhancing mass in the right kidney, multiple pulmonary nodules, and left pleural thickening with large pleural effusion. Pleura biopsy revealed a malignant neoplasm composed of cells with predominantly clear cytoplasm. Considering the large mass in the right kidney, clear cell renal cell carcinoma (RCC) was the main differential diagnosis. The diagnosis in this case was not definitive by histology alone since clear cell RCC markers such as RCC and AE1/AE3 were negative, and CD10 was only focally positive. Transcription factor E3 (TFE3) immunohistochemistry was positive, while the XP11.2 translocation testing was negative. Electron microscopy demonstrated that the tumor cells had abundant cytoplasmic glycogen and lipid, focal long microvilli lining rare lumina, and adjacent interdigitating cell membranes joining the neoplastic cells, indicating a diagnosis of renal clear cell carcinoma. In addition, numerous crystalline-like dense granules were identified in the cytoplasm of the neoplastic cells, which are reminiscent of those typically seen in alveolar soft part sarcoma and rarely described in XP11.2 translocation RCC. Overall, this renal tumor likely represents a variant of XP11.2 translocation RCC, overexpressing TFE3 with dense granules.

Genomic instability in human cancer: Molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition.

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology.

Histiocytic sarcoma as a secondary malignancy: pathobiology, diagnosis, and treatment.

Histiocytic sarcoma (HS) is an extremely rare non-Langerhans cell disorder with an aggressive course and limited treatment options. Recent advances in molecular/genetic sequencing have suggested a common clonal origin between various hematolymphoid disorders and cases of secondary HS. Deriving conclusions from previously reported cases of HS arising secondarily to certain hematolymphoid disorders, here we have tried to provide insight into the mechanisms influencing this evolution. We also discuss a clinical case of a 72-year-old man with a diagnosis of chronic myeloid leukemia (CML), presenting subsequently with a heterogeneous liver mass positive with a diagnosis of HS. The liver mass showed a retained BCR-ABL1 translocation suggesting clonality between the CML and HS. As seen in our case and other reported cases of HS derived secondarily, the concurrent expression of immunoglobulin heavy (IGH)-/light-chain rearrangements or cytogenetic markers common to the primary malignancy suggests an evolutionary mechanism involving lineage switching that could potentially be influenced by genetic or epigenetic cues which may occur at the level of a progenitor or the malignant cell itself.

Metastatic Ovarian Serous Adenocarcinoma Clinically Presenting as Inflammatory Breast Cancer.

Metastatic disease to the breast is a rare event, accounting for 0.5-2% of all breast cancers. Outside of metastases from the contralateral breast, malignant ovarian epithelial tumors are the most common origin of these metastases. Here, we present a very rare case of a high-grade ovarian serous adenocarcinoma presenting clinically as inflammatory breast cancer in a 70-year-old woman.

A Primary Telangiectatic Mandibular Osteosarcoma With Germ-Line Malignancy-Associated DNA Damage Repair Gene Polymorphisms: A Case Report.

Primary mandibular telangiectatic osteosarcomas are very rare lesions, with only nine cases reported. Histologically, these lesions show multiple cystic blood-filled cavities traversed by neoplastic bone in septa lined by high-grade malignant cells. Here, we report an 81-year-old woman who presented with a mandibular mass, which was surgically resected and analyzed by histologic examination and whole exome DNA sequencing. A diagnosis of telangiectatic osteosarcoma was given. Comparative sequencing data analysis of paired benign and tumor DNA revealed 1577 variants unique to the tumor DNA, which clustered into several gene families, including those regulating DNA repair and apoptosis. Comparison of benign and tumor DNA revealed many shared gene polymorphisms associated with an increased cancer risk. These included polymorphisms in the ATM, p53, BRCA1, and BRCA2 and many other genes. Interestingly, the patient's family history showed an unusually high cancer incidence, likely related to these cancer risk-associated polymorphisms. To our knowledge, this is the first-time sequencing applied to a mandibular telangiectatic osteosarcoma. Our findings may shed light on the molecular origins of these rare tumors and how they may relate to other tumors in related kindreds.

Sulfide regulation of cardiovascular function in health and disease.

Hydrogen sulfide (H2S) has emerged as a gaseous signalling molecule with crucial implications for cardiovascular health. H2S is involved in many biological functions, including interactions with nitric oxide, activation of molecular signalling cascades, post-translational modifications and redox regulation. Various preclinical and clinical studies have shown that H2S and its synthesizing enzymes - cystathionine γ-lyase, cystathionine ß-synthase and 3-mercaptosulfotransferase - can protect against cardiovascular pathologies, including arrhythmias, atherosclerosis, heart failure, myocardial infarction and ischaemia-reperfusion injury. The bioavailability of H2S and its metabolites, such as hydropersulfides and polysulfides, is substantially reduced in cardiovascular disease and has been associated with single-nucleotide polymorphisms in H2S synthesis enzymes. In this Review, we highlight the role of H2S, its synthesizing enzymes and metabolites, their roles in the cardiovascular system, and their involvement in cardiovascular disease and associated pathologies. We also discuss the latest clinical findings from the field and outline areas for future study.

A Rare Case of Pure Primary Large Cell Neuroendocrine Carcinoma of the Gallbladder.

Primary large cell neuroendocrine carcinoma (LCNEC) of the gallbladder is a rare malignancy which is often associated with non-LCNEC histologic components. Histologically "pure" LCNECs of the gallbladder are exceedingly rare with only 15 cases reported in the medical literature. Clinically, LCNECs present with abdominal pain and jaundice and follow an aggressive course with patients surviving a median of 15 months following initial diagnosis. To our knowledge, we present the 16th case of a histologically pure LCNEC in a 62-year-old African American male who was successfully treated surgically. After discharge, he was subsequently lost to follow-up. Due to the extreme rarity of such disease entity, accurate diagnosis and proper management are essential for the best clinical outcome.

The ataxia-telangiectasia mutated gene product regulates the cellular acid-labile sulfide fraction.

The ataxia-telangiectasia mutated (ATM) protein regulates cell cycle checkpoints, the cellular redox state, and double-stranded DNA break repair. ATM loss causes the disorder ataxia-telangiectasia (A-T), distinguished by ataxia, telangiectasias, dysregulated cellular redox and iron responses, and an increased cancer risk. We examined the sulfur pool in A-T cells, with and without an ATM expression vector. While free and bound sulfide levels were not changed with ATM expression, the acid-labile sulfide faction was significantly increased. ATM expression also increased cysteine desulfurase (NFS1), NFU1 iron-sulfur cluster scaffold homolog protein, and several mitochondrial complex I proteins' expression. Additionally, ATM expression suppressed cystathionine ß-synthase and cystathionine γ-synthase protein expression, cystathionine γ-synthase enzymatic activity, and increased the reduced to oxidized glutathione ratio. This last observation is interesting, as dysregulated glutathione is implicated in A-T pathology. As ATM expression increases the expression of proteins central in initiating 2Fe-2S and 4Fe-4S cluster formation (NFS1 and NFU1, respectively), and the acid-labile sulfide faction is composed of sulfur incorporated into Fe-S clusters, our data indicates that ATM regulates aspects of Fe-S cluster biosynthesis, the transsulfuration pathway, and glutathione redox cycling. Thus, our data may explain some of the redox- and iron-related pathologies seen in A-T.

CircP4HB regulates ferroptosis via SLC7A11-mediated glutathione synthesis in lung adenocarcinoma.

Background: Circular ribonucleic acids (circRNAs) play a key role in the development of different types of cancer. Ferroptosis is a type of programmed cell death that contributes to cancer progression. However, the role of circRNAs in lung adenocarcinoma (LUAD) ferroptosis remains unclear. Methods: The gene expression levels of circRNA P4HB (circP4HB), microRNA-1184 (miR-1184) and Solute carrier family 7 member 11 (Slc7a11), also known as Xct were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Ferroptosis of established LUAD cells was induced by erastin. Cell viability was examined via Cell Counting Kit 8 assays. Ferroptosis was evaluated by malondialdehyde (MDA), Prostaglandin-endoperoxide Synthase 2 (Ptgs2), lipid reactive oxygen species (lipid ROS), and JC-1 detection. The mechanism of circP4HB/miR-1184/SLC7A11 was investigated by luciferase reporter assays, RNA immunoprecipitation, RNA pull-down, and western blot assays. A functional for circP4HB in vivo was determined using xenograft nude mice models. Results: CircP4HB expression levels were increased in LUAD. It triggered glutathione (GSH) synthesis and, therefore protected LUAD cells from ferroptosis induced by erastin. CircP4HB may function as a competing endogenous RNA by modulating miR-1184 to regulate SLC7A11. CircP4HB inhibited ferroptosis by regulating miR-1184/ SLC7A11-mediated GSH synthesis. In vivo, overexpression of circP4HB promoted tumor growth and inhibited ferroptosis. Conclusions: The circRNA, circP4HB acts as a novel ferroptosis suppressor in LUAD. Furthermore, circP4HB protects LUAD from ferroptosis via modulation of the miR-1184/SLC7A11 axis. Our findings identified circP4HB as a novel biomarker in LUAD and warrants further investigation in the early diagnosis and treatment of LUAD.

Methamphetamine causes cardiovascular dysfunction via cystathionine gamma lyase and hydrogen sulfide depletion.

Methamphetamine (METH) is an addictive illicit drug used worldwide that causes significant damage to blood vessels resulting in cardiovascular dysfunction. Recent studies highlight increased prevalence of cardiovascular disease (CVD) and associated complications including hypertension, vasospasm, left ventricular hypertrophy, and coronary artery disease in younger populations due to METH use. Here we report that METH administration in a mouse model of 'binge and crash' decreases cardiovascular function via cystathionine gamma lyase (CSE), hydrogen sulfide (H2S), nitric oxide (NO) (CSE/H2S/NO) dependent pathway. METH significantly reduced H2S and NO bioavailability in plasma and skeletal muscle tissues co-incident with a significant reduction in flow-mediated vasodilation (FMD) and blood flow velocity revealing endothelial dysfunction. METH administration also reduced cardiac ejection fraction (EF) and fractional shortening (FS) associated with increased tissue and perivascular fibrosis. Importantly, METH treatment selectively decreased CSE expression and sulfide bioavailability along with reduced eNOS phosphorylation and NO levels. Exogenous sulfide therapy or endothelial CSE transgenic overexpression corrected cardiovascular and associated pathological responses due to METH implicating a central molecular regulatory pathway for tissue pathology. These findings reveal that therapeutic intervention targeting CSE/H2S bioavailability may be useful in attenuating METH mediated cardiovascular disease.

Primary Pulmonary Leiomyosarcoma in a Forty-Year-Old Woman.

Primary pulmonary leiomyosarcomas (PPLs) are rare aggressive malignancies originating from the smooth muscle cells of the pulmonary interstitium, bronchial tree, or blood vessels. Accounting for <0.5% of lung tumors, PPLs are often initially undetected or misdiagnosed as pulmonary emboli, cardiac neoplasms, or as other more common lung cancer subtypes. Due to their aggressive and often lethal clinical profile, the diagnostic delay of PPL can significantly affect patient outcomes and must be avoided. Here we describe a case of PPL in a 40-year-old woman.

Bad Smells and Broken DNA: A Tale of Sulfur-Nucleic Acid Cooperation.

Hydrogen sulfide (H2S) is a gasotransmitter that exerts numerous physiologic and pathophysiologic effects. Recently, a role for H2S in DNA repair has been identified, where H2S modulates cell cycle checkpoint responses, the DNA damage response (DDR), and mitochondrial and nuclear genomic stability. In addition, several DNA repair proteins modulate cellular H2S concentrations and cellular sulfur metabolism and, in turn, are regulated by cellular H2S concentrations. Many DDR proteins are now pharmacologically inhibited in targeted cancer therapies. As H2S and the enzymes that synthesize it are increased in many human malignancies, it is likely that H2S synthesis inhibition by these therapies is an underappreciated aspect of these cancer treatments. Moreover, both H2S and DDR protein activities in cancer and cardiovascular diseases are becoming increasingly apparent, implicating a DDR-H2S signaling axis in these pathophysiologic processes. Taken together, H2S and DNA repair likely play a central and presently poorly understood role in both normal cellular function and a wide array of human pathophysiologic processes. Here, we review the role of H2S in DNA repair.

Ethylmalonic Encephalopathy 1 Protein Is Increased in Colorectal Adenocarcinoma.

BACKGROUND/AIM: Ethylmalonic encephalopathy 1 protein (ETHE1) plays an important role in sulfide catabolism and polysulfide formation. As sulfides and polysulfides have recently been identified as playing important roles in cancer, we hypothesized that ETHE1 expression would be increased in colon cancer. MATERIALS AND METHODS: We used tissue microarray analysis to compare ETHE1 expression in benign colonic epithelium compared to colonic adenocarcinoma. In total, 26 benign colonic epithelial samples were compared to 122 cases of colonic adenocarcinomas. RESULTS: Compared to benign colonic epithelium, ETHE1 expression was significantly increased (~two-fold) in colonic adenocarcinoma. Additionally, this expression increased with increasing colon cancer tumor grades. CONCLUSION: ETHE1 expression is increased in colon cancer compared to benign colonic epithelium. These data, combined with previous studies, suggest that ETHE1 may contribute to colon carcinogenesis by promoting tumor cell bioenergetics and polysulfide formation.

Cystathionine Gamma-Lyase Is Increased in Testicular Seminomas, Embryonal, and Yolk Sac Tumors.

BACKGROUND: Testicular cancer constitutes 1.0% of male cancer and typically carries a good prognosis. As far as we are aware, the role for hydrogen sulfide in testicular cancer and the level of hydrogen sulfide-synthesizing enzyme have never been addressed. Here we examined cystathionine gamma-lyase (CSE) expression in several germ-cell testicular tumors. MATERIALS AND METHODS: Tissue microarrays were employed to examine CSE expression in 32 benign testicular samples, 88 testicular seminomas, 34 embryonal carcinomas, 4 mature teratomas, and 16 yolk sac tumors, and CSE expression was compared to that seen in benign testicular tissue. RESULTS: Compared to benign testicular tissue, CSE expression was increased in all three types of testicular neoplasm but not in mature teratomas. Highest CSE expression was identified in embryonal carcinomas, which often show a relatively aggressive clinical course. CONCLUSION: For the first time, we show that CSE is increased in several common testicular germ-cell tumor types.

Molecular Functions of Hydrogen Sulfide in Cancer.

Hydrogen sulfide (H2S) is a gasotransmitter that exerts a multitude of functions in both physiologic and pathophysiologic processes. H2S-synthesizing enzymes are increased in a variety of human malignancies, including colon, prostate, breast, renal, urothelial, ovarian, oral squamous cell, and thyroid cancers. In cancer, H2S promotes tumor growth, cellular and mitochondrial bioenergetics, migration, invasion, angiogenesis, tumor blood flow, metastasis, epithelia-mesenchymal transition, DNA repair, protein sulfhydration, and chemotherapy resistance Additionally, in some malignancies, increased H2S-synthesizing enzyme expression correlates with a worse prognosis and a higher tumor stage. Here we review the role of H2S in cancer, with an emphasis on the molecular mechanisms by which H2S promotes cancer development, progression, dedifferentiation, and metastasis.

Synchronous Occurrence of Splenic Pleomorphic Mantle Cell Lymphoma and Esophageal Adenocarcinoma with Overexpression of BCL1 Protein.

Synchronous occurrences of mantle cell lymphoma (MCL), or intermediate lymphocytic lymphoma, and other malignancies are rare. Such cases present diagnostic and especially therapeutic challenges, making them of particular interest to study. We report a case of synchronic MCL and an esophageal tumor in an elderly male patient. Morphologically, the tumors were classified as splenic pleomorphic MCL and adenocarcinoma of the esophagus occurring concurrently. The pleomorphic MCL mimicked diffuse large B cell lymphoma (DLBCL) but lacked larger centroblast- or immunoblast-like cells. Curiously, both tumors overexpressed cyclin D1 by immunohistochemistry. This is an important feature that distinguishes MCL pathologically from two of its closest entities in the differential diagnosis: chronic lymphocytic leukemia and DLBCL, the latter of which mantle cells cannot transform into. The lymphoproliferation revealed IGH/CCND1 translocation by FISH, but the esophageal adenocarcinoma only showed CCND1 aneuploidy without break-apart signals. Since the gastrointestinal (GI) tract is a common site of extranodal involvement by MCL and lymphomatous polyposis can present as GI polyps, adequate care was taken to differentiate the esophageal adenocarcinoma from advanced stagings of MCL, as well as metastatic adenocarcinoma. Despite numerous immunohistochemical stainings studied, only BCL1 was demonstrated to have partial overlap in both tumors. The patient underwent esophagectomy and splenectomy. A subsequent metastatic primary lung squamous cell carcinoma was diagnosed, after which the patient expired. MCL typically presents at an advanced stage and has been deemed incurable with a prognosis of only several years. It is unclear whether the patient succumbed to complications of his MCL or the metastatic squamous cell carcinoma. Furthermore, he was lost to follow-up for a year and only received treatment after his third cancer was diagnosed. We have reviewed previous reports of synchronic mantle cell lymphoma and other solid tumors or hematological malignancies in the literature.

Plasmablastic Lymphoma, a Rare Entity in Bone Marrow with Unusual Immunophenotype and Challenging Differential Diagnosis.

Plasmablastic lymphoma (PBL) is an aggressive malignancy that usually occurs in the setting of immunosuppression. The immunohistochemical profile of PBL is that of terminally differentiated B lymphocytes. CD138, CD38, and MUM1 are usually immunopositive. However, pan B-cell markers such as CD20 and PAX-5 are usually negative. MYC rearrangement is the most commonly encountered genetic alteration, with immunoglobulin (IG), especially immunoglobulin heavy (IGH) chain, being the most frequent partner. We report a case of PBL in a 48-year-old human immunodeficiency virus- (HIV-) positive male who was admitted to the hospital with signs and symptoms suspicious for tumor lysis syndrome. Bone marrow examination revealed hypercellular marrow with trilineage hypoplasia and sheets of intermediate to large neoplastic cells with basophilic vacuolated cytoplasm comprising the majority of cellular elements of the bone marrow. The neoplastic cells were negative for conventional B-cell, T-cell, plasma cell, and myeloid markers, while flow cytometric analysis revealed an abnormal CD45-dim population that was partially weakly positive for CD71 and CD79b. The diagnosis was initially thought to be a high-grade primitive hematopoietic neoplasm, possibly an acute undifferentiated leukemia. BOB-1, however, was immunopositive in the neoplastic cells, confirming its B-cell origin. MYC was positive by immunohistochemistry and break-apart FISH, as were CD45, MUM-1, and EMA immunostains. There was immunoglobulin kappa (IGK) light chain gene rearrangement by polymerase chain reaction (PCR). Additionally, Epstein-Barr virus- (EBV-) encoded small RNAs (EBER) were positive by in situ hybridization (ISH). The tumor proliferation index by Ki-67 immunostaining approached 95%. Although the tumor cells were negative for CD38 and CD138, the diagnosis of PBL was still rendered. We recommend using a broad spectrum of B-cell markers, including BOB-1 and OCT-2, in such challenging cases of B-cell lymphomas with no expression of conventional B-cell markers. We also emphasize that the negative CD38 and CD138 should not exclude PBL from the differential diagnosis.

The Ataxia telangiectasia-mutated and Rad3-related protein kinase regulates cellular hydrogen sulfide concentrations.

The ataxia telangiectasia-mutated and Rad3-related (ATR) serine/threonine kinase plays a central role in the repair of replication-associated DNA damage, the maintenance of S and G2/M-phase genomic stability, and the promotion of faithful mitotic chromosomal segregation. A number of stimuli activate ATR, including persistent single-stranded DNA at stalled replication folks, R loop formation, hypoxia, ultraviolet light, and oxidative stress, leading to ATR-mediated protein phosphorylation. Recently, hydrogen sulfide (H2S), an endogenous gasotransmitter, has been found to regulate multiple cellular processes through complex redox reactions under similar cell stress environments. Three enzymes synthesize H2S: cystathionine-ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Since H2S can under some conditions cause DNA damage, we hypothesized that ATR activity may regulate cellular H2S concentrations and H2S-syntheszing enzymes. Here we show that human colorectal cancer cells carrying biallelic knock-in hypomorphic ATR mutations have lower cellular H2S concentrations than do syngeneic ATR wild-type cells, and all three H2S-synthesizing enzymes show lower protein expression in the ATR hypomorphic mutant cells. Additionally, ATR serine 428 phosphorylation is altered by H2S donor and H2S synthesis enzyme inhibition, while the oxidative-stress induced phosphorylation of the ATR-regulated protein CHK1 on serine 345 is increased by H2S synthesis enzyme inhibition. Lastly, inhibition of H2S production potentiated oxidative stress-induced double-stranded DNA breaks in the ATR hypomorphic mutant compared to ATR wild-type cells. Our findings demonstrate that the ATR kinase regulates and is regulated by H2S.

Hydrogen Sulfide and Hydrogen Sulfide-Synthesizing Enzymes Are Altered in a Case of Oral Adenoid Cystic Carcinoma.

Adenoid cystic carcinomas (ACC) constitute 1% of all head and neck malignancies and are very rare in the oral cavity. With < 60 oral ACCs described, their pathobiology is incompletely understood. Here, we report a case of oral cavity ACC in a 54-year-old woman. Since recent studies have demonstrated that several human tumors overexpress the hydrogen sulfide (H2S)-synthesizing enzymes cystathionine-ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST), and also show dysregulated H2S levels, we examined these biomarkers in the oral ACC and compared the results to those of adjacent benign oral epithelium. Western blotting was used to compare the protein expression of CBS, CSE, 3-MST, nicotinamide phosphoribosyl transferase, and mitoNEET in ACC and adjacent benign oral mucosae. High-performance liquid chromatography was used to quantify the differences in tissue H2S concentrations between the two biopsy types. We found that all the proteins examined here were increased in the ACC compared to adjacent benign oral mucosae. Interestingly, H2S concentrations were decreased approximately 30% in ACC compared to benign mucosae. Thus, in one example of this rare tumor type, the enzymes that synthesize H2S are increased, while tissue H2S levels are lower than those found in adjacent benign oral mucosae. Although limited to a single rare tumor type, to our knowledge this is the second time H2S concentrations have been directly quantified inside a human tumor. Last, our results may indicate that alterations in H2S synthesis and metabolism may be important in the pathobiology of ACC.