Cannabidiol‐induced activation of the metallothionein pathway impedes anticancer effects of disulfiram and its metabolite CuET

Disulfiram (DSF), an established alcohol‐aversion drug, is a candidate for repurposing in cancer treatment. DSF’s antitumor activity is supported by preclinical studies, case reports, and small clinical trials; however, ongoing clinical trials of advanced‐stage cancer patients encounter variable results. Here, we show that one reason for the inconsistent clinical effects of DSF may reflect interference by other drugs. Using a high‐throughput screening and automated microscopy, we identify cannabidiol, an abundant component of the marijuana plant used by cancer patients to mitigate side effects of chemotherapy, as a likely cause of resistance to DSF. Mechanistically, in cancer cells, cannabidiol triggers the expression of metallothioneins providing protective effects by binding heavy metal‐based substances including the bis‐diethyldithiocarbamate‐copper complex (CuET). CuET is the documented anticancer metabolite of DSF, and we show here that the CuET’s anticancer toxicity is effectively neutralized by metallothioneins. Overall, this work highlights an example of undesirable interference between cancer therapy and the concomitant usage of marijuana products. In contrast, we report that insufficiency of metallothioneins sensitizes cancer cells toward CuET, suggesting a potential predictive biomarker for DSF repurposing in oncology.     

Long-term productive human cytomegalovirus infection of a human neuroblastoma cell line

Human neuroblastoma cell line UKF-NB-4 persistently infected with human cytomegalovirus (HCMV) strain AD169 was established to study the effects of long-term HCMV infection on virus production and phenotypic characteristics of tumour cells. The cells designated UKF-NB-4^AD169 were subcultured (80 subcultures) over a period of more than 2 years after initiation of infection. UKF-NB-4^AD169 cells continued to produce infectious virus in successive passages, with a titre ranging from 9 × 10³ to 1 × 10⁵ and from 2 × 10¹ to 2 × 10² plaque-forming units per 10⁶ cells and 1 ml culture medium, respectively; 10–20% of the cells produced HCMV-specific antigens, while 6–13% produced infectious virus progeny. The number of HCMV-specific DNA copies ranged from 9 × 10⁴ to 9 × 10⁶ per 10⁶ cells. Transmission electron microscopy confirmed the productive nature of HCMV infection. UKF-NB-4^AD169 cultures proliferated, with population doubling time ranging from 24.5 to 26.6 hr (19.5 to 20.3 hr for UKF-NB-4) and cell viability from 79% to 85% (91–96% for UKF-NB-4). Significantly lower amounts of tyrosine hydroxylase and decreased activity for dopamine-β-hydroxylase than in uninfected cells were observed in UKF-NB-4^AD169 cells. However, the expression of N-myc oncoprotein was significantly increased in persistently infected cultures. Our results show that long-term productive HCMV infection of UKF-NB-4 cell line is associated with the modulation of phenotypic properties, which may be related to the biological behaviour of neuroblastoma cells. © 1996 Wiley-Liss, Inc.     

Psychiatric disorders and stages of smoking.

BACKGROUND: We examined the role of DSM-III-R psychiatric disorders in predicting the subsequent onset of daily smoking, smokers' progression to nicotine dependence, and the persistence of smoking. METHODS: The Tobacco Supplement of the National Comorbidity Survey was administered to a representative subsample of 4414 persons 15-54 years of age. DSM-III-R psychiatric disorders and information on age of onset of psychiatric disorders, daily smoking, and smoking cessation were ascertained with the World Health Organization's Composite International Diagnostic Interview. RESULTS: Preexisting psychiatric disorders that have not remitted (i.e., active disorders) predicted an increased risk for the first onset of daily smoking and for smokers' progression to nicotine dependence. The increased risk applied across most of the disorders examined in the study, including major depression, anxiety disorders, and substance use disorders. Persons with four or more active disorders were at higher risk for daily smoking (2.1 vs. 1.4) and for nicotine dependence (2.9 vs. 1.4) than were persons with one active disorder. With few exceptions, remitted (i.e., past) disorders did not predict the subsequent onset of daily smoking. Preexisting psychiatric disorders did not influence smokers' potential for quitting; the persistence of smoking in the year preceding the interview was unrelated to history of psychiatric disorders. CONCLUSIONS: The results suggest the possibility of additional and previously unrecognized public health benefits of early treatment of mental disorders, in that persons with various mental disorders whose illness had remitted were not at increased risk for daily smoking, in contrast with persons with active disorders.     

A suction-curet apparatus for endometrial biopsy

A suction-curet apparatus for endometrial biopsy is described. The primary application of the apparatus is in the determination of ovulation in cases of infertility. An electric motor suction apparatus is employed instead of suction by syringe. The curved tube cannula is preferred to the straight instrument, and can usually be inserted without dilation of the cervix. The method can also be used for diagnosis of adenocarcinoma of the uterus, hyperplasia of the endometrium, the study of indocrinopathic amenorrhea, and for obtaining fertilized ovum at very early stages of implantation.     

Survival after liver transplantation in the United States: a disease-specific analysis of the UNOS database.

Our goal was to describe disease-specific survival and the clinical variables that predict survival in a large national cohort of adult liver transplant recipients. Data on 17,044 adult patients who received an initial orthotopic liver transplant between 1990 and 1996 with follow-up through 1999 was obtained from the United Network for Organ Sharing (UNOS). Disease-specific Kaplan-Meier survival plots and Cox Proportional Hazards models were estimated, and differences in the clinical characteristics of patients at the time of transplantation by disease were examined. Overall posttransplant survival currently exceeds 85% in the first year and is approaching 75% at 5 years. Unadjusted Kaplan-Meier survival is improved for recipients who are younger, female, and in better clinical condition. Survival is a function of disease and level of illness: cancer, fulminant liver failure, alcoholic liver disease, and the hepatitidies have the poorest prognosis, while primary billiary cirrohsis and sclerosing cholangitis have the best. Recipients who were outpatients before transplantation have longer survival than those transplanted from the hospital or intensive care unit. Although the model for end-stage liver disease (MELD) score was designed to predict pretransplant survival, patients with higher MELD scores have poorer posttransplant survival, but the MELD score is less predictive than the specific disease. Differences in disease-specific survival are partially explained by differences in disease severity at the time of transplantation. In conclusion, Disease-specific survival models indicate that there remains tremendous variability in survival as a function of underlying liver disease. However, a significant portion of the difference in survival between diseases arises from differences in clinical characteristics at the time of transplantation.     

Calcium influx pathways in rat pancreatic ducts

A number of agonists increase intracellular Ca²⁺ activity, [Ca²⁺]_i, in pancreatic ducts, but the influx/efflux pathways and intracellular Ca²⁺ stores in this epithelium are unknown. The aim of the present study was to characterise the Ca²⁺ influx pathways, especially their pH sensitivity, in native pancreatic ducts stimulated by ATP and carbachol, CCH. Under control conditions both agonists led to similar changes in [Ca²⁺]_i. However, these Ca²⁺ transients, consisting of peak and plateau phases, showed different sensitivities to various experimental manoeuvres. In extracellular Ca²⁺-free solutions, the ATP-induced [Ca²⁺]_i peak decreased by 25%, but the CCH-induced peak was unaffected; both plateaus were inhibited by 90%. Flufenamate inhibited the ATP-induced peak by 35%, but not the CCH-evoked peak; the plateaus were inhibited by 75–80%. La³⁺ inhibited the ATP-induced plateau fully, but that induced by CCH by 55%. In resting ducts, an increase in extracellular pH, pH_e, by means of HEPES and HCO₃ ⁻/CO₂ buffers, increased [Ca²⁺]_i; a decrease in pH_e had the opposite effect. In stimulated ducts the pH-evoked effects on Ca²⁺ influx were more pronounced and depended on the agonist used. At pH_e 6.5 both ATP- and CCH-evoked plateaus were inhibited by about 50%. At pH 8.0 the ATP-stimulated plateau was inhibited by 27%, but that stimulated by CCH was increased by 72%. Taken together, we show that CCH stimulates Ca²⁺ release followed by Ca²⁺ influx that is moderately sensitive to flufenamate, La³⁺, depolarisation, it is inhibited by low pH, but stimulated by high pH. ATP stimulates Ca²⁺ release and probably an early Ca²⁺ influx, which is more markedly sensitive to flufenamate and La³⁺, and is both inhibited by low and high pH. Thus our study indicates that there are at least two separate Ca²⁺ influx pathways in pancreatic ducts cells. Received: 4 December 1995/Received after revision and accepted: 1 February 1996     

Difference between the tau protein of Alzheimer paired helical filament core and normal tau revealed by epitope analysis of monoclonal antibodies 423 and 7.51.

The microtubule-associated protein tau that is incorporated into paired helical filaments (PHFs) undergoes some form of aberrant posttranslational processing in Alzheimer disease. Difficulties in deciding which changes are critical for PHF formation stem in part from the lack of immunochemical markers specific for PHF tau. The only monoclonal antibody (mAb) that is known to react with PHF tau but not with the predominant normal adult tau species is mAb 423. Another mAb (7.51, described in this paper) recognizes a segment of tau that is included in the minimal recognition unit required by mAb 423. Unlike 423, which is PHF tau-specific, mAb 7.51 recognizes all PHF core-derived tau as well as native soluble tau and recombinant tau expressed in bacteria and so serves as a generic tau marker. Both epitopes are in the 12-kDa fragment released from the Pronase-resistant core of the PHF (which encompasses the tandem repeat region). The mAb 7.51 epitope requires segments located in the last two repeats, which are common to all tau isoforms. The mAb 423 epitope requires sequences located near both the N and the C terminus of the 12-kDa fragment common to three- and four-repeat tau isoforms. Fragments denatured by concentrated formic acid and SDS regain 423 reactivity when denaturing agents are removed. Since the primary amino acid sequences of PHF tau and normal tau are identical in the repeat region, we conclude that 423 reactivity also requires a modification(s) occurring within an approximately 90-residue segment that are not present in tau proteins so far described in the human brain.