The emerging SARS-CoV-2 papain-like protease: Its relationship with recent coronavirus epidemics.

The papain-like protease (PLpro ) is an important enzyme for coronavirus polyprotein processing, as well as for virus-host immune suppression. Previous studies reveal that a molecular analysis of PLpro indicates the catalytic activity of viral PLpro and its interactions with ubiquitin. By using sequence comparisons, molecular models, and protein-protein interaction maps, PLpro was compared in the three recorded fatal CoV epidemics, which involved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome CoV (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV). The pairwise sequence comparison of SARS-CoV-2 PLpro indicated similarity percentages of 82.59% and 30.06% with SARS-CoV PLpro and MERS-CoV PLpro , respectively. In comparison with SARS-CoV PLpro , in SARS-CoV-2, the PLpro had a conserved catalytic triad of C111, H278, and D293, with a slightly lower number of polar interface residues and of hydrogen bonds, a higher number of buried interface sizes, and a lower number of residues that interact with ubiquitin and PLpro . These features might contribute to a similar or slightly lower level of deubiquitinating activity in SARS-CoV-2 PLpro. It was, however, a much higher level compared to MERS-CoV, which contained amino acid mutations and a low number of polar interfaces. SARS-CoV-2 PLpro and SARS-CoV PLpro showed almost the same catalytic site profiles, interface area compositions and polarities, suggesting a general similarity in deubiquitination activity. Compared with MERS-CoV, SARS-CoV-2 had a higher potential for binding interactions with ubiquitin. These estimated parameters contribute to the knowledge gap in understanding how the new virus interacts with the immune system.

Three species of Echinococcus granulosus sensu lato infect camels on the Arabian Peninsula.

We report on the genetic identity of 36 Echinococcus cysts that were collected during a recent slaughterhouse survey of 810 locally bred camels (dromedaries) in the Eastern Province of the Kingdom of Saudi Arabia. Analysis of a partial nad1 gene sequence showed that the majority (n = 29) belonged to E. granulosus sensu stricto, four to E. canadensis G6/7, and three to E. ortleppi. Eight of the 29 E. granulosus s.s. cysts contained protoscoleces; all other cysts were calcified and non-viable. This is the first report of the presence E. ortleppi from the Arabian Peninsula, a parasite that is typically transmitted via cattle. The results indicate widespread infection of camels with CE in eastern Saudi Arabia and an active role of camels in the lifecycles of at least E. granulosus s.s.. Complete cox1 haplotype analysis of 21 E. granulosus s.s. isolates shows that the majority of variants circulating in eastern Saudi Arabia is distinct from but closely related to haplotypes from neighboring countries in the Middle East, which indicates the presence of this parasite in KSA for a longer period of time. All isolates of E. granulosus s.s. in this study belonged to the G1 cluster, although the G3 genotype has previously also been reported from the Middle East.

From SARS and MERS CoVs to SARS-CoV-2: Moving toward more biased codon usage in viral structural and nonstructural genes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging disease with fatal outcomes. In this study, a fundamental knowledge gap question is to be resolved by evaluating the differences in biological and pathogenic aspects of SARS-CoV-2 and the changes in SARS-CoV-2 in comparison with the two prior major COV epidemics, SARS and Middle East respiratory syndrome (MERS) coronaviruses. METHODS: The genome composition, nucleotide analysis, codon usage indices, relative synonymous codons usage, and effective number of codons (ENc) were analyzed in the four structural genes; Spike (S), Envelope (E), membrane (M), and Nucleocapsid (N) genes, and two of the most important nonstructural genes comprising RNA-dependent RNA polymerase and main protease (Mpro) of SARS-CoV-2, Beta-CoV from pangolins, bat SARS, MERS, and SARS CoVs. RESULTS: SARS-CoV-2 prefers pyrimidine rich codons to purines. Most high-frequency codons were ending with A or T, while the low frequency and rare codons were ending with G or C. SARS-CoV-2 structural proteins showed 5 to 20 lower ENc values, compared with SARS, bat SARS, and MERS CoVs. This implies higher codon bias and higher gene expression efficiency of SARS-CoV-2 structural proteins. SARS-CoV-2 encoded the highest number of over-biased and negatively biased codons. Pangolin Beta-CoV showed little differences with SARS-CoV-2 ENc values, compared with SARS, bat SARS, and MERS CoV. CONCLUSION: Extreme bias and lower ENc values of SARS-CoV-2, especially in Spike, Envelope, and Mpro genes, are suggestive for higher gene expression efficiency, compared with SARS, bat SARS, and MERS CoVs.

Molecular characterization of fowl aviadenoviruses species D and E associated with inclusion body hepatitis in chickens and falcons indicates possible cross-species transmission.

During the period from 2015 to 2017, frequent outbreaks of inclusion body hepatitis (IBH) were observed in broiler chickens and falcons in Saudi Arabia. Fifty samples were collected from both species. The histopathological examination and polymerase chain reaction confirmed the IBH infection in eight samples (five samples from chickens and three samples from falcons). The genomic sequence and phylogenetic analysis based on nucleotide and amino acid sequences of Saudi strains, reference fowl aviadenoviruses (FAdVs) and field viruses available in Genbank revealed that all investigated FAdVs clustered into FAdV-2 (species D) and FAdV-6 (species E). The host-dependent characterization revealed that falcon origin strains showed low identity (∼35%) with falcon adenoviruses isolated from USA, which clustered into a separate group. The identification of FAdV-D and FAdV-E in diseased falcons and chickens indicates cross-species transmission although falcons and chickens are phylogenetically different. The control of IBH infection in falcons and chickens should be based on the separation of carriers and susceptible chickens as well as falcons to prevent cross-species contact. Vaccination is an important method for prevention of IBH. The characterization of newly emerging FAdV strains provides valuable information for the development of an efficacious control strategy based on the molecular structure of current circulating FAdV strains in different species of birds.

Honey feeding protects kidney against cisplatin nephrotoxicity through suppression of inflammation.

Cisplatin is a highly effective chemotherapeutic drug used to treat a wide variety of solid tumors. However, its use was limited due its dose-limiting toxicity to the kidney. Currently, there are no therapies available to treat or prevent cisplatin nephrotoxicity. Honey is a naturally occurring complex liquid and widely used in traditional Ayurvedic medicine to treat many illnesses. However, its effect on cisplatin nephrotoxicity is unknown. To determine the role of honey in cisplatin nephrotoxicity, animals were pretreated orally for a week and then cisplatin was administered. Honey feeding was continued for another 3 days. Our results show that animals with cisplatin-induced kidney dysfunction, as determined by increased serum creatinine, which received honey feeding had less kidney dysfunction. Improved kidney function was associated with better preservation of kidney morphology in honey-treated group as compared to the cisplatin alone-treated group. Interestingly, honey feeding significantly reduced cisplatin-induced tubular epithelial cell death, immune infiltration into the kidney as well as cytokine and chemokine expression and excretion as compared to cisplatin treated animals. Western blot analysis shows that cisplatin-induced increase in phosphorylation of NFkB was completely suppressed with honey feeding. In conclusion, honey feeding protects the kidney against cisplatin nephrotoxicity through suppression of inflammation and NFkB activation.

Effects of aflatoxin B1 on human breast cancer (MCF-7) cells: cytotoxicity, oxidative damage, metabolic, and immune-modulatory transcriptomic changes.

Aflatoxin B1 (AFB1) is a potent mycotoxin that is commonly produced by molds such as Aspergillus (A.) flavus and A. parasiticus. AFB1 is associated with several health adverse effects in humans including mutagenesis and carcinogenesis. Aflatoxin is commonly secreted in the milk leading to deleterious effects on breast tissue and potential nursing infants. However, the effects of aflatoxins, particularly AFB1, on the breast cells are less investigated. In this study, AFB1-associated effects on human breast cancer cell lines (MCF-7) were investigated. AFB1 caused significant cytotoxicity on MCF-7 cells. Such cytotoxicity had a positive correlation with the induction of oxidative stress. In addition, AFB1 caused significant transcriptomic alterations in xenobiotics and drug-metabolizing enzymes, transporters, and antioxidant enzymes. Besides, AFB1 upregulated pro-inflammatory markers such as tumor necrosis factor-α and cyclooxygenase-2 with a significant reduction of mRNA expressions of the immunity-related genes including interleukins 8 and 10.

Maxillary neoplasms in four dromedary camels.

Four camels (Camelus dromedarius) presented to the Veterinary Teaching Hospital at King Faisal University with maxillary masses. On radiographs, the masses were multicystic and expanded the maxillary bone. The tumors were diagnosed by histopathologic examination as conventional ameloblastoma, two cases as intraosseous squamous cell carcinoma, and central odontogenic fibroma with ossification. To the authors' knowledge, this is the first report of ameloblastoma in a camel, the first detailed description of maxillary squamous cell carcinoma in camels, and the first report of central odontogenic fibroma in any animal species.

Prevalence and Antimicrobial Susceptibility of Campylobacter Species with Particular Focus on the Growth Promoting, Immunostimulant and Anti-Campylobacter jejuni Activities of Eugenol and Trans-Cinnamaldehyde Mixture in Broiler Chickens.

Campylobacter species (spp.) are one of the most important causes of human bacterial gastroenteritis in foods of animal origin. Recently, with the spread of multi-drug-resistant (MDR) and extensively drug-resistant (XDR) Campylobacter spp., natural alternative therapeutic methods are urgently required. Phytogenic active principles have gained considerable attention due to their proficiency to enhance gut health and, thereby, performance of broiler chickens. Thus, the current study aims to determine the prevalence and antimicrobial resistance of Campylobacter spp. of different chicken sources in Sharkia Governorate, Egypt, and to assess the growth-promoting, immunostimulant and antimicrobial effects of a mixture of eugenol and trans-cinnamaldehyde in an in vivo approach. A total of 101 (67.3%) campylobacter isolates was identified, according to both phenotypic and genotypic techniques. Moreover, all of the campylobacter isolates were resistant to erythromycin, trimethoprim/sulfamethoxazole, and ampicillin (100% each). Of note, a dietary supplementation of the mixture of eugenol and trans-cinnamaldehyde led to a significant improvement of the feed conversion ratio and body weight gain and a decrease in the cecal C. jejuni loads in the broilers challenged with XDR C. jejuni. Additionally, eugenol and the trans-cinnamaldehyde mixture had protective activities via the down-regulation of XDR C. jejuni (flaA, virB11 and wlaN) virulence genes and proinflammatory cytokines (TNF-α, IL-2, IL-6, and IL-8), and the up-regulation of anti-inflammatory cytokine IL-10. Thus, we recommend the usage of a mixture of eugenol and trans-cinnamaldehyde as an alternative to antimicrobials for the control and treatment of campylobacter infections.

Hepatic lobe torsion in 3 dromedary camels.

Hepatic lobe torsion is a rare condition in domestic animals. Clinical signs are variable, with some cases remaining subclinical and others resulting in death. Most cases are diagnosed either by laparotomy or during postmortem examination. During postmortem inspection of 670 slaughtered dromedary camels, hepatic lobe torsion of the quadrate lobe was detected in 3 adult female camels. Clinical signs had not been noted on antemortem veterinary inspection, and hepatic lobe torsion was likely an incidental finding. Histologically, the affected liver lobe exhibited severe hepatocellular loss with replacement by fibrous connective tissue. When investigating abdominal pain in camels, veterinarians should include hepatic lobe torsion in the list of differential diagnoses.

Content of total aflatoxin, lead, and cadmium in the bovine meat and edible offal: study of their human dietary intake, health risk assessment, and molecular biomarkers.

The objectives of the present study were first to determine the residual contents of total aflatoxins (AFTs), lead (Pb), and cadmium (Cd) in the edible tissues of the cattle reared in Al-Ahsa, Saudi Arabia. Al-Ahsa is the largest governorate in the Eastern Province of Saudi Arabia. The two main economic activities of Al-Ahsa are oil production (industrial) and agriculture. Besides, dietary intake and possible health risks for Saudi population were further calculated. In order to establish potential molecular biomarkers for xenobiotic exposure in cattle, the mRNA expression of xenobiotic-metabolizing enzymes (XMEs) including cytochrome P450 (CYP) 1A1, NAD(P)H dehydrogenase [quinone] 1 (NQO1), metallothionein (MT) 1A, and heat shock protein (HSP) 70 was investigated in the different tissues of the cattle. The tested XMEs were selected because of their specific roles in the metabolism and detoxification of AFTs, Pb, and Cd. The obtained results revealed that the liver had significantly the highest AFT content, while all examined muscle samples had no AFT residues. Consumption of the bovine liver and kidneys represents the highest source for the dietary exposure to total AFTs (0.05-0.98 µg/kg/day), Pb (0.06-0.19 mg/kg/day), and Cd (0.08-0.19 mg/kg/day) among the examined tissues. Therefore, excessive intake of such organs might pose a public health concern, particularly among children. Significant upregulation of mRNA expressions of CYP1A1, NQO1, MT1A, and HSP70 was observed in the different tissues of the cattle in comparison with the muscle. This upregulation had significant positive correlation with the accumulated AFTs, Pb, and Cd. This indicates the possible use of CYP1A1, NQO1, MT1A, and HSP70 as potential biomarkers for the exposure of the cattle to AFTs, Pb, and Cd.

Repurposing of FDA-approved antivirals, antibiotics, anthelmintics, antioxidants, and cell protectives against SARS-CoV-2 papain-like protease.

SARS-CoV-2 or Coronavirus disease 19 (COVID-19) is a rapidly spreading, highly contagious, and sometimes fatal disease for which drug discovery and vaccine development are critical. SARS-CoV-2 papain-like protease (PLpro) was used to virtually screen 1697 clinical FDA-approved drugs. Among the top results expected to bind with SARS-CoV-2 PLpro strongly were three cell protectives and antioxidants (NAD+, quercitrin, and oxiglutatione), three antivirals (ritonavir, moroxydine, and zanamivir), two antimicrobials (doripenem and sulfaguanidine), two anticancer drugs, three benzimidazole anthelmintics, one antacid (famotidine), three anti-hypertensive ACE receptor blockers (candesartan, losartan, and valsartan) and other miscellaneous systemically or topically acting drugs. The binding patterns of these drugs were superior to the previously identified SARS CoV PLpro inhibitor, 6-mercaptopurine (6-MP), suggesting a potential for repurposing these drugs to treat COVID-19. The objective of drug repurposing is the rapid relocation of safe and approved drugs by bypassing the lengthy pharmacokinetic, toxicity, and preclinical phases. The ten drugs with the highest estimated docking scores with favorable pharmacokinetics were subjected to molecular dynamics (MD) simulations followed by molecular mechanics/generalized Born surface area (MM/GBSA) binding energy calculations. Phenformin, quercetin, and ritonavir all demonstrated prospective binding affinities for COVID-19 PLpro over 50 ns MD simulations, with binding energy values of -56.6, -40.9, and -37.6 kcal/mol, respectively. Energetic and structural analyses showed phenformin was more stable than quercetin and ritonavir. The list of the drugs provided herein constitutes a primer for clinical application in COVID-19 patients and guidance for further antiviral studies.Communicated by Ramaswamy H. Sarma.

Effect of curcumin and Meriva on the lung metastasis of murine mammary gland adenocarcinoma.

BACKGROUND: Curcumin is one of the most studied natural compounds which has been used as a feed additive for centuries. Curcumin exhibits low oral bioavailability in rodents and human. Curcumin formulated with phosphatidylcholine (Meriva) increases curcumin bioavailability five-fold compared to original curcumin. The aim of this study was to evaluate the efficacy of curcumin conjugated with phosphatidylcholine as an anticancer agent. MATERIALS AND METHODS: In this xenograft study, mammary gland tumor cell line (ENU1564) was inoculated into the mammary fat pad of athymic nude mice. The mice were treated orally with either curcumin or Meriva. The tumor and its lung metastasis were evaluated grossly, microscopically, and immunohistochemically. RESULTS: Meriva significantly reduced the expression of MMP-9 and lung metastasis of our cell line used in this experimental model. CONCLUSION: Curcumin conjugated with phosphatidylcholine increased the efficacy of curcumin as an anticancer agent.

Nephroprotective Effects of Benzyl Isothiocyanate and Resveratrol Against Cisplatin-Induced Oxidative Stress and Inflammation.

This study was performed to compare the nephroprotective effects of benzyl isothiocyanate (BITC) and resveratrol (RES) and investigate the nephroprotective efficacy of their combination against cisplatin-induced acute renal injury. Five animal groups (each of eight) received either normal saline, a single intraperitoneal injection of cisplatin (20 mg/kg) at the sixth day, cisplatin plus oral RES (30 mg/kg) or BITC (100 mg/kg in diet), or a combination of both for 10 days. Compared to saline-treated mice, cisplatin-intoxicated mice had significantly higher (p < 0.05) serum levels of urea, creatinine, interleukin-1ß (IL-1ß), and tumor necrosis factor-α. Moreover, biochemical analysis of kidney tissue homogenates showed that cisplatin intoxication was associated with significantly higher (p < 0.05) tissue levels of malondialdehyde (MDA) and lower levels of reduced glutathione and activities of endogenous antioxidant enzymes (glutathione peroxidase, superoxide dismutase, and catalase) in comparison to normal controls. Histopathological and immunohistochemical examinations of renal tissue slices from cisplatin-intoxicated mice showed interstitial leukocytic infiltration, tortuous tubules with vacuolated epithelium, luminal casts, and overexpression of cyclooxygenase-II enzyme. On the other hand, treatment with RES or BITC ameliorated all the previous parameters. The effects of both compounds were comparable in all assessed parameters, except IL-1ß serum concentration and renal tissue MDA concentration (which were significantly lower in the RES group). Interestingly, treatment with BITC and RES combination restored the normal concentrations of all the aforementioned biochemical parameters, as well as near normal histological and immunohistochemical pictures. In conclusion, BITC exerted nearly comparable nephroprotective, antioxidant, and anti-inflammatory effects to RES and the combination of both agents showed more potent nephroprotective effects against cisplatin than each one alone.

Foot-and-mouth disease virus O/ME-SA/Ind 2001 lineage outbreak in vaccinated Holstein Friesian cattle in Saudi Arabia in 2016.

BACKGROUND: Foot-and-mouth disease virus (FMDV) is a highly contagious viral infection of large ruminants. Despite the massive application of vaccines against FMDV, several outbreaks are still being reported in Africa and Asia. AIM: To perform molecular characterization of FMDV in an outbreak among a cattle herd Saudi Arabia in 2016. This herd had been vaccinated with a polyvalent FMDV vaccine. METHODS: To investigate this outbreak, we collected specimens from 77 animals showing typical clinical signs of FMDV. Specimens including sera, nasal swabs, and tissues (tongue, coronary bands, hooves, and hearts) were collected. We tested the collected cattle sera for the presence of FMDV antibodies with commercial ELISA kits. In addition, we tested the swabs for the presence of the most common FMDV strains (O, A, Asia-1 and SAT-2) with RT-PCR using serotype-specific oligonucleotides. RESULTS: Serology showed that 22% of the tested sera were positive. Molecular testing of the examined swabs confirmed that 24% of the tested animals were positive. Our sequencing analysis confirmed that the circulating strains of FMDV belonged to FMDV serotype O. The phylogenetic tree based on the FMDV-VP-1 gene revealed high nucleotide identity between the circulating strains and the Bangladesh strain (99%). These strains were distinct (shared 89% nucleotide identity) from the FMDV-O strains used for the preparation of the vaccine administered to the animals in this herd. Moreover, they had 7% nucleotide difference between the FMDV-O strains reported in Saudi Arabian in 2013. CONCLUSION: More in-depth molecular characterization of these FMDV strains is warranted.

Nephroprotective efficacy of ceftriaxone against cisplatin-induced subchronic renal fibrosis in rats.

Cisplatin, or cis-diamminedichloridoplatinum(II), (CDDP) is a broad-spectrum antineoplastic chemotherapeutic agent with a potent efficacy against several malignancies. Its main clinical antineoplastic therapy-limiting adverse effect is nephrotoxicity, where the developments of effective nephroprotectors are needed. Therefore, the present study aimed to investigate the nephroprotective and antifibrotic potential of ceftriaxone (CTX) against CDDP-induced toxicity. Male Wister rats were treated with saline or CTX (100 or 200 mg kg-1 bw) an hour before CDDP administration (1 mg kg-1 bw). All the treatments were intraperitoneally administered twice weekly for consecutive 10 weeks. Twenty-four hours after last CDDP dose, blood samples were collected, then the animals were euthanized and their kidneys were isolated for measurements. CDDP significantly increased serum uric acid, urea, and creatinine contents. Toxicopathic changes showed that CDDP induced marked tubulointerstitial damage, overexpressed fibrogenic factors α-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1), and down expressed cellular proliferating biomarker bromodeoxyuridine (BrdU). CTX pretreatment, particularly 200 mg/kg bw, improved the renal function biomarkers; histoarchitecture; and α-SMA, TGF-ß1, and BrdU expressions. It could be concluded that CTX is endowed with antifibrotic properties and could be, therefore, used as adjuvant therapy to improve CDDP-induced nephrotoxicity. Further clinical researches are necessary to evaluate whether CTX may exhibit a new therapeutic choice for treating renal fibrotic diseases.

Celecoxib aggravates cardiac apoptosis in L-NAME-induced pressure overload model in rats: Immunohistochemical determination of cardiac caspase-3, Mcl-1, Bax and Bcl-2.

The mechanism of celecoxib cardiovascular adverse events was earlier investigated; yet in-depth investigations are needed to assess the involvement of its pro-apoptotic effect throughout this process. An in-vivo chronic rat model of pressure overload employing NÊ·-nitro-l-arginine methyl ester (L-NAME) was tested at different time intervals to ensure the occurrence of persistent myocardial apoptosis along with pressure overload. Seven groups of male Wistar rats were assigned as (i) distilled water; (ii-iv) L-NAME (60 mg/kg) for 6, 12 or 16 weeks; (v-vii) L-NAME [16 weeks] + celecoxib (25, 50 or 100 mg/kg), from week 13 to week 16. Treatment with L-NAME for 6, 12 or 16 weeks increased systolic blood pressure, serum level of creatine kinase-MB and lactate dehydrogenase. Further, it induced cardiac hypertrophy, detected in terms of greater heart weight index and cardiomyocyte cross-sectional area and produced interstitial and perivascular fibrosis. Moreover, administration of L-NAME increased cardiac immunostaining for activated caspase-3 and Bax/Bcl-2 ratio whereas; immunostaining for Mcl-1 was decreased. Administration of celecoxib (25, 50 or 100 mg/kg) aggravated the L-NAME-induced toxicity. The work results shed the light on the putative pro-apoptotic effect of celecoxib at a risk state of pressure overload comparable to the clinical condition of essential hypertension.

Nephroprotective effect of bee honey and royal jelly against subchronic cisplatin toxicity in rats.

Cisplatin is one of the most potent and effective chemotherapeutic agents. However, its antineoplastic use is limited due to its cumulative nephrotoxic side effects. Therefore, the present study was undertaken to examine the nephroprotective potential of dietary bee honey and royal jelly against subchronic cisplatin toxicity in rats. Male Wistar rats were randomly divided into controls, cisplatin-treated, bee honey-pretreated cisplatin-treated and royal jelly-pretreated cisplatin-treated groups. Bee honey and royal jelly were given orally at doses of 20 and 100 mg/kg, respectively. Subchronic toxicity was induced by cisplatin (1 mg/kg bw, ip), twice weekly for 10 weeks. Cisplatin treated animals revealed a significant increase in serum level of renal injury products (urea, creatinine and uric acid). Histopathologically, cisplatin produced pronounced tubulointerstitial injuries, upregulated the fibrogenic factors, α-smooth muscle actin (α-SMA) and transforming growth factor ß1(TGF-ß1), and downregulated the cell proliferation marker, bromodeoxyuridine (Brdu). Dietary bee honey and royal jelly normalized the elevated serum renal injury product biomarkers, improved the histopathologic changes, reduced the expression of α-SMA and TGF-ß1 and increased the expression of Brdu. Therefore, it could be concluded that bee honey, and royal jelly could be used as dietary preventive natural products against subchronic cisplatin-induced renal injury.

Curcumin induces apoptosis in a murine mammary gland adenocarcinoma cell line through the mitochondrial pathway.

Curcumin, a phenol in turmeric (Curcuma longa), has been studied for the last decade as a potential anticancer drug. It has been shown to reduce viability of the highly malignant, metastatic rat mammary gland cell line ENU1564 in culture and reduce metastasis of these cells injected into nude mice. The purpose of this study was to identify the mechanisms by which curcumin induces apoptosis in these ENU1564 cells in vitro, and to examine its effects on mitochondrial membrane potential and mitochondrial Ca(2+) homeostasis. The results show that curcumin induced apoptosis in ENU1564 cells through the intrinsic pathway of apoptosis, as evident by an increase in mitochondrial Ca(2+) accumulation and a decrease in mitochondrial membrane potential. However, treatment of the ENU1564 cells with the mitochondrial uniporter inhibitor RU-360 prior to curcumin treatment partially inhibited the curcumin effects. SKF-96365, a store-operated Ca(2+) channel blocker, suppressed the curcumin effect on mitochondrial Ca(2+). In addition, curcumin down-regulated the expressions of Bcl-2 and procaspase-3 and increased the production of reactive oxygen species in ENU1564 cells. These data suggest that the mitochondrial Ca(2+) is the leading factor by which curcumin induced apoptosis in ENU1564 cells, followed by reactive oxygen species production and inhibition of Bcl-2 oncoprotein.