Targeting Cancer Lysosomes with Good Old Cationic Amphiphilic Drugs.

Being originally discovered as cellular recycling bins, lysosomes are today recognized as versatile signaling organelles that control a wide range of cellular functions that are essential not only for the well-being of normal cells but also for malignant transformation and cancer progression. In addition to their core functions in waste disposal and recycling of macromolecules and energy, lysosomes serve as an indispensable support system for malignant phenotype by promoting cell growth, cytoprotective autophagy, drug resistance, pH homeostasis, invasion, metastasis, and genomic integrity. On the other hand, malignant transformation reduces the stability of lysosomal membranes rendering cancer cells sensitive to lysosome-dependent cell death. Notably, many clinically approved cationic amphiphilic drugs widely used for the treatment of other diseases accumulate in lysosomes, interfere with their cancer-promoting and cancer-supporting functions and destabilize their membranes thereby opening intriguing possibilities for cancer therapy. Here, we review the emerging evidence that supports the supplementation of current cancer therapies with lysosome-targeting cationic amphiphilic drugs.

Bile Acid Diarrhea Is Associated With an Increased Incidence of Gastrointestinal Cancers.

INTRODUCTION: Bile acid diarrhea (BAD) is an underrecognized and socially debilitating disease caused by high concentrations of bile acids in the colon. Bile acids directly and indirectly promote carcinogenesis. In this article, we investigated whether individuals with BAD have an increased risk of gastrointestinal (GI) cancers. METHODS: By using the Danish health registries, adult individuals with BAD were identified by International Classification of Diseases 10th revision code K90.8 or referral to the diagnostic 75selenium-homotaurocholic acid test followed by prescription of a bile acid sequestrant within 365 days (n = 5,245). Age- and sex-matched individuals without BAD were included for comparison (n = 52,450). We analyzed the cumulative incidence of GI cancers after BAD diagnosis and the odds ratios (ORs) of GI cancer 8 and 15 years before BAD diagnosis/matching. RESULTS: Cumulative incidence of GI cancer 6 years after BAD diagnosis/matching was 1.6% in the BAD group and 1.1% in controls ( P = 0.01). The ORs of total GI cancer 8 and 15 years before BAD diagnosis were 6.16 (5.08-7.48) and 5.19 (4.28-6.29), respectively. Furthermore, 47 individuals with BAD (0.9%) and 250 (0.5%) controls died of GI cancer. DISCUSSION: This nationwide cohort study indicates an association between BAD and GI cancers. We found both a higher incidence of GI cancer after BAD diagnosis compared with controls and increased OR of GI cancer before BAD diagnosis. Bearing in mind the underdiagnosis of BAD, the delay of BAD diagnosis, and the carcinogenic effect of bile acids, these findings warrant further investigations of the risk of GI cancer in individuals with BAD.

Elucidating the glucose-lowering effect of the bile acid sequestrant sevelamer.

AIM: Bile acid sequestrants are cholesterol-lowering drugs, which also improve glycaemic control in people with type 2 diabetes. The mechanism behind the glucose-lowering effect is unknown but has been proposed to be mediated by increased glucagon-like peptide-1 (GLP-1) secretion. Here, we investigated the glucose-lowering effects of sevelamer including any contribution from GLP-1 in people with type 2 diabetes. MATERIALS AND METHODS: In a randomized, double-blind, placebo-controlled, crossover study, 15 people with type 2 diabetes on metformin monotherapy underwent two 17-day treatment periods with the bile acid sequestrant sevelamer and placebo, respectively, in a randomized order and with an interposed wash-out period of minimum 6 weeks. On days 15 and 17 of each treatment period, participants underwent experimental days with 4-h liquid meal tests and application of concomitant infusion of exendin(9-39)NH2 or saline. RESULTS: Compared with placebo, sevelamer improved insulin sensitivity (assessed by homeostatic model assessment of insulin resistance) and beta-cell sensitivity to glucose and lowered fasting and postprandial plasma glucose concentrations. In both treatment periods, exendin(9-39)NH2 increased postprandial glucose excursions compared with saline but without absolute or relative difference between the two treatment periods. In contrast, exendin(9-39)NH2 abolished the sevelamer-induced improvement in beta-cell glucose sensitivity. CONCLUSIONS: The bile acid sequestrant sevelamer improved insulin sensitivity and beta-cell sensitivity to glucose, but using the GLP-1 receptor antagonist exendin(9-39)NH2 we were not able to detect a GLP-1-mediated glucose-lowering effect of sevelamer in individuals with type 2 diabetes. Nevertheless, the sevelamer-induced improvement of beta-cell sensitivity to glucose was shown to be GLP-1-dependent.

The impact of type 2 diabetes and glycaemic control on mortality and clinical outcomes in hospitalized patients with COVID-19 in the capital region of Denmark.

AIM: To explore the impact of type 2 diabetes (T2D), glycaemic control and use of glucose-lowering medication on clinical outcomes in hospitalized patients with COVID-19. MATERIALS AND METHODS: For all patients admitted to a hospital in the Capital Region of Denmark (1 March 2020 to 1 December 2021) with confirmed COVID-19, we extracted data on mortality, admission to intensive care unit (ICU), demographics, comorbidities, medication use and laboratory tests from the electronic health record system. We compared patients with T2D to patients without diabetes using Cox proportional hazards models adjusted for available confounding variables. Outcomes were 30-day mortality and admission to an ICU. For patients with T2D, we also analysed the association of baseline haemoglobin A1c (HbA1c) levels and use of specific glucose-lowering medications with the outcomes. RESULTS: In total, 4430 patients were analysed, 1236 with T2D and 2194 without diabetes. The overall 30-day mortality was 19% (n = 850) and 10% (n = 421) were admitted to an ICU. Crude analyses showed that patients with T2D both had increased mortality [hazard ratio (HR) 1.37; 95% CI 1.19-1.58] and increased risk of ICU admission (HR 1.28; 95% CI 1.04-1.57). When adjusted for available confounders, this discrepancy was attenuated for both mortality (adjusted HR 1.13; 95% CI 0.95-1.33) and risk of ICU admission (adjusted HR 1.01; 95% CI 0.79-1.29). Neither baseline haemoglobin A1c nor specific glucose-lowering medication use were significantly associated with the outcomes. CONCLUSION: Among those hospitalized for COVID-19, patients with T2D did not have a higher risk of death and ICU admission, when adjusting for confounders.

Comment on Yang et al. Bile Acid Diarrhea: From Molecular Mechanisms to Clinical Diagnosis and Treatment in the Era of Precision Medicine. Int. J. Mol. Sci. 2024, 25, 1544.

We have with great interest read the recent review on the molecular mechanisms underlying bile acid diarrhea (BAD) by Yang et al [...].

Serum lipidome unravels a diagnostic potential in bile acid diarrhoea.

OBJECTIVE: Bile acid diarrhoea (BAD) is debilitating yet treatable, but it remains underdiagnosed due to challenging diagnostics. We developed a blood test-based method to guide BAD diagnosis. DESIGN: We included serum from 50 treatment-naive patients with BAD diagnosed by gold standard 75selenium homotaurocholic acid test, 56 feature-matched controls and 37 patients with non-alcoholic fatty liver disease (NAFLD). Metabolomes were generated using mass spectrometry covering 1295 metabolites and compared between groups. Machine learning was used to develop a BAD Diagnostic Score (BDS). RESULTS: Metabolomes of patients with BAD significantly differed from controls and NAFLD. We detected 70 metabolites with a discriminatory performance in the discovery set with an area under receiver-operating curve metric above 0.80. Logistic regression modelling using concentrations of decanoylcarnitine, cholesterol ester (22:5), eicosatrienoic acid, L-alpha-lysophosphatidylinositol (18:0) and phosphatidylethanolamine (O-16:0/18:1) distinguished BAD from controls with a sensitivity of 0.78 (95% CI 0.64 to 0.89) and a specificity of 0.93 (95% CI 0.83 to 0.98). The model was independent of covariates (age, sex, body mass index) and distinguished BAD from NAFLD irrespective of fibrosis stage. BDS outperformed other blood test-based tests (7-alpha-hydroxy-4-cholesten-3-one and fibroblast growth factor 19) currently under development. CONCLUSIONS: BDS derived from serum metabolites in a single-blood sample showed robust identification of patients with BAD with superior specificity and sensitivity compared with current blood test-based diagnostics.

The bile-gut axis and metabolic consequences of cholecystectomy.

Cholelithiasis and cholecystitis affect individuals of all ages and are often treated by surgical removal of the gallbladder (cholecystectomy), which is considered a safe, low-risk procedure. Nevertheless, recent findings show that bile and its regulated storage and excretion may have important metabolic effects and that cholecystectomy is associated with several metabolic diseases postoperatively. Bile acids have long been known as emulsifiers essential to the assimilation of lipids and absorption of lipid-soluble vitamins, but more recently, they have also been reported to act as metabolic signaling agents. The nuclear receptor, farnesoid X receptor (FXR), and the G protein-coupled membrane receptor, Takeda G protein-coupled receptor 5 (TGR5), are specific to bile acids. Through activation of these receptors, bile acids control numerous metabolic functions. Cholecystectomy affects the storage and excretion of bile acids, which in turn may influence the activation of FXR and TGR5 and their effects on metabolism including processes leading to metabolic conditions such as metabolic dysfunction-associated steatotic liver disease and metabolic syndrome. Here, with the aim of elucidating mechanisms behind cholecystectomy-associated dysmetabolism, we review studies potentially linking cholecystectomy and bile acid-mediated metabolic effects and discuss possible pathophysiological mechanisms behind cholecystectomy-associated dysmetabolism.

Expression of bile acid receptors and transporters along the intestine of patients with type 2 diabetes and controls.

CONTEXT: The enterohepatic circulation of bile acids depends on intestinal absorption by bile acid transporters and activation of bile acid receptors, which stimulates secretion of hormones regulating glucose and lipid metabolism and appetite. Distribution of bile acid transporters and receptors in the human gut and their potential involvement in type 2 diabetes (T2D) pathophysiology remain unknown. OBJECTIVE: We explored the expression of genes involved in bile acid metabolism throughout the intestines of patients with T2D and matched healthy controls. METHODS: Intestinal mucosa biopsies sampled along the intestinal tract in 12 individuals with T2D and 12 healthy controls were subjected to mRNA sequencing. We report expression profiles of apical sodium-dependent bile acid transporter (ASBT), organic solute transporter (OST) α/ß, farnesoid X receptor (FXR), Takeda G receptor 5 (TGR5), fibroblast growth factor 19 (FGF19) and FGF receptor 4 (FGFR4). RESULTS: Expression of ASBT and OSTα/ß was evident in the duodenum of both groups with increasing levels through the small intestine, and no (ASBT) or decreasing levels (OSTα/ß) through the large intestine. The FXR expression pattern followed that of OSTα/ß whereas FGFR4 were evenly expressed through the intestines. Negligible levels of TGR5 and FGF19 were evident. Patients with T2D exhibited lower levels of FGF19, FXR, ASBT and OSTα/ß mRNAs compared with healthy controls, although the differences were not statistically significant after adjusting for multiple testing. CONCLUSIONS: We demonstrate distinct expression patterns of bile acid transporters and receptors through the intestinal tract with signs of reduced ASBT, OSTα/ß, FXR and FGF19 mRNAs in T2D.

Gluco-metabolic response to exogenous oxytocin in totally pancreatectomized patients and healthy individuals.

Oxytocin has been proposed to possess glucose-stabilizing effects through the release of insulin and glucagon from the pancreas. Also, exogenous oxytocin has been shown to stimulate extrapancreatic glucagon secretion in depancreatized dogs. Here, we investigated the effect of exogenous oxytocin on circulating levels of pancreatic and gut-derived glucose-stabilizing hormones (insulin [measured as C-peptide], glucagon, glucagon-like peptide 1 [GLP-1], and glucose-dependent insulinotropic polypeptide). We studied nine pancreatectomized (PX) patients and nine healthy controls (CTRLs) (matched on age and body mass index) before, during, and after an intravenous infusion of 10 IU of oxytocin administered over 12 min. Oxytocin did not increase plasma glucagon levels, nor induce any changes in plasma glucose, C-peptide, or GIP in any of the groups. Oxytocin decreased plasma glucagon levels by 19 ± 10 % in CTRLs (from 2.0 ± 0.5 [mean ± SEM] to 1.3 ± 0.2 pmol/l, P = 0.0025) and increased GLP-1 by 42 ± 22 % in PX patients (from 9.0 ± 1.0-12.7 ± 1.0 pmol/l, P = 0.0003). Fasting plasma glucose levels were higher in PX patients compared with CTRLs (13.1 ± 1.1 vs. 5.1 ± 0.1 mmol/l, P < 0.0001). In conclusion, the present findings do not support pancreas-mediated glucose-stabilizing effects of acute oxytocin administration in humans and warrant further investigation of oxytocin's gluco-metabolic effects.

Exogenous glucagon-like peptide 2 counteracts exogenous cholecystokinin-induced gallbladder contraction in healthy men.

BACKGROUND AND OBJECTIVE: Studies in humans and mice have demonstrated that the gut hormone glucagon-like peptide 2 (GLP-2) promotes gallbladder relaxation and refilling. Here, we assessed the effect of exogenous GLP-2 on gallbladder motility in the fasted state of healthy men with and without infusion of the potent gallbladder-contracting hormone cholecystokinin (CCK). METHODS: In a randomized, double-blind, placebo-controlled, crossover study, 15 male participants (mean [SD]: age 24.7 [3.6] years; body mass index 22.9 [1.6] kg/m2) underwent four experimental days receiving two infusions on each day: either CCK (0.4 pmol × kg-1 × min-1, time 0-180 min) + GLP-2 (10 pmol × kg-1 × min-1, time 30-240 min), CCK + placebo, placebo + GLP-2, or placebo + placebo, respectively. Gallbladder volume was measured at baseline and throughout the 4-hour study day using ultrasonography. RESULTS: Compared to placebo + placebo, GLP-2 + placebo did not affect gallbladder volume, but when infused in combination with CCK, GLP-2 completely abolished the strong gallbladder-contracting effect seen during CCK + placebo infusion, restoring baseline levels of gallbladder volume. CONCLUSION: Exogenous GLP-2 counteracts exogenous CCK-induced gallbladder emptying in healthy men, pointing to a possible therapeutic potential for GLP-2 as a relaxing modulator of gallbladder smooth muscle tone (e.g., as bridge to surgery in biliary colic). The effect may also explain the gallbladder-related adverse events reported for GLP-2 receptor agonists used in the treatment of short bowel syndrome.

Bile acid diarrhoea.

Bile acid diarrhoea is a socially debilitating disease caused by irritation of the colonic mucosa due to a spillover of bile acids from the small intestine into the colon. Studies estimate a prevalence of 1-2% of the adult population, but many patients never seek help or are misdiagnosed. Bile acid diarrhoea is treated with bile acid sequestrants, however, new research shows superior effect on reported symptoms with the glucagon-like peptide 1 receptor agonist liraglutide.

The Epidemiology of Bile Acid Diarrhea in Denmark.

Objective: Bile acid diarrhea (BAD) is a socially debilitating disease with frequent bowel movements, urgency, and fecal incontinence as the main symptoms. It is caused by excessive bile acid levels in the colon and is most commonly treated with bile acid sequestrants. It is estimated that 1-2% of the population suffers from the disease, but only a fraction of these are properly diagnosed with the gold standard 75selenium-homotaurocholic acid (SeHCAT) test. Here, we use nationwide registries to describe the demographic characteristics of individuals suffering from BAD in Denmark. Methods: Since the International Classification of Diseases diagnosis code for BAD was not used until 2021, we identified the BAD population by referral to SeHCAT testing followed by a prescription of a bile acid sequestrant (colestyramine, colestipol or colesevelam) within 365 days. The study period was from 2003 to 2021. Results: During the study period, a total of 5264 individuals with BAD were identified with large differences between the five regions in Denmark. The number of prescriptions of colestyramine and colesevelam, the number of SeHCAT tests, and the number of individuals diagnosed with BAD increased during the study period. The BAD population had more co-morbidities and more health care contacts as well as lower levels of education and income compared with age- and sex-matched controls from the general population. Conclusion: Using the Danish registries, we identified a BAD population, which seems to be inferior in health care and socio-economic parameters compared with the Danish general population.

MYC mRNA expression throughout the intestine is not associated with body mass index or type 2 diabetes.

Inspired by the prospect of intestinal MYC reduction as a putative drug target against metabolic disorders, we investigated MYC mRNA expression in intestinal mucosa biopsies sampled using double-balloon enteroscopy along the entire intestinal tract of 12 patients with type 2 diabetes and 12 matched healthy controls. In these individuals with body mass index (BMI) ranging from 20 to 31 kg/m2 , the mean MYC mRNA expression varied along the intestine with the lowest level observed at the distal small intestine and the greatest expression levels in the proximal small intestine and in the colon. We do not see a correlation between intestinal mucosal MYC mRNA expression and BMI or glycaemic control, and thus, our findings do not support the hypothesis of intestinal MYC as a putative drug target against obesity and metabolic diseases.

Protocol for a 30-day randomised, parallel-group, non-inferiority, controlled trial investigating the effects of discontinuing renin-angiotensin system inhibitors in patients with and without COVID-19: the RASCOVID-19 trial.

INTRODUCTION: The COVID-19 pandemic caused by the virus SARS-CoV has spread rapidly and caused damage worldwide. Data suggest a major overrepresentation of hypertension and diabetes among patients experiencing severe courses of COVID-19 including COVID-19-related deaths. Many of these patients receive renin-angiotensin system (RAS) inhibiting therapy, and evidence suggests that treatment with angiotensin II receptor blockers (ARBs) could attenuate SARS-CoV-induced acute respiratory distress syndrome, and ACE inhibitors and ARBs have been suggested to alleviate COVID-19 pulmonary manifestations. This randomised clinical trial will address whether RAS inhibiting therapy should be continued or discontinued in hospitalised patients with COVID-19. METHODS AND ANALYSIS: This trial is a 30-day randomised parallel-group non-inferiority clinical trial with an embedded mechanistic substudy. In the main trial, 215 patients treated with a RAS inhibitor will be included. The participants will be randomly assigned in a 1:1 ratio to either discontinue or continue their RAS inhibiting therapy in addition to standard care. The patients are included during hospitalisation and followed for a period of 30 days. The primary end point is number of days alive and out of hospital within 14 days after recruitment. In a mechanistic substudy, 40 patients treated with RAS inhibition, who are not in hospital and not infected with COVID-19 will be randomly assigned to discontinue or continue their RAS inhibiting therapy with the primary end point of serum ACE2 activity. ETHICS AND DISSEMINATION: This trial has been approved by the Scientific-Ethical Committee of the Capital Region of Denmark (identification no. H-20026484), the Danish Medicines Agency (identification no. 2020040883) and by the Danish Data Protection Agency (P-2020-366). The results of this project will be compiled into one or more manuscripts for publication in international peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: 2020-001544-26; NCT04351581.

Enterohepatic, Gluco-metabolic, and Gut Microbial Characterization of Individuals With Bile Acid Malabsorption.

Background and Aims: Bile acid malabsorption (BAM) is a debilitating disease characterized by loose stools and high stool frequency. The pathophysiology of BAM is not well-understood. We investigated postprandial enterohepatic and gluco-metabolic physiology, as well as gut microbiome composition and fecal bile acid content in patients with BAM. Methods: Twelve participants with selenium-75 homocholic acid taurine test-verified BAM and 12 healthy controls, individually matched on sex, age, and body mass index, were included. Each participant underwent 2 mixed meal tests (with and without administration of the bile acid sequestrant colesevelam) with blood sampling and evaluation of gallbladder motility; bile acid content and microbiota composition were evaluated in fecal specimens. Results: Patients with BAM were characterized by increased bile acid synthesis as assessed by circulating 7-alpha-hydroxy-4-cholesten-3-one, fecal bile acid content, and postprandial concentrations of glucose, insulin, C-peptide, and glucagon. The McAuley index of insulin sensitivity was lower in patients with BAM than that in healthy controls. In patients with BAM, colesevelam co-administered with the meal reduced postprandial concentrations of bile acids and fibroblast growth factor 19 and increased 7-alpha-hydroxy-4-cholesten-3-one concentrations but did not affect postprandial glucagon-like peptide 1 responses or other gluco-metabolic parameters. Patients with BAM were characterized by a gut microbiome with low relative abundance of bifidobacteria and high relative abundance of Blautia, Streptococcus, Ruminococcus gnavus, and Akkermansia muciniphila. Conclusion: Patients with BAM are characterized by an overproduction of bile acids, greater fecal bile acid content, and a gluco-metabolic profile indicative of a dysmetabolic prediabetic-like state, with changes in their gut microbiome composition potentially linking their enterohepatic pathophysiology and their dysmetabolic phenotype. ClinicalTrials.gov number NCT03009916.

Repurposing Cationic Amphiphilic Antihistamines for Cancer Treatment.

Non-small cell lung cancer (NSCLC) is one of the deadliest cancers worldwide. In search for new NSCLC treatment options, we screened a cationic amphiphilic drug (CAD) library for cytotoxicity against NSCLC cells and identified several CAD antihistamines as inducers of lysosomal cell death. We then performed a cohort study on the effect of CAD antihistamine use on mortality of patients diagnosed with non-localized cancer in Denmark between 1995 and 2011. The use of the most commonly prescribed CAD antihistamine, loratadine, was associated with significantly reduced all-cause mortality among patients with non-localized NSCLC or any non-localized cancer when compared with use of non-CAD antihistamines and adjusted for potential confounders. Of the less frequently described CAD antihistamines, astemizole showed a similar significant association with reduced mortality as loratadine among patients with any non-localized cancer, and ebastine use showed a similar tendency. The association between CAD antihistamine use and reduced mortality was stronger among patients with records of concurrent chemotherapy than among those without such records. In line with this, sub-micromolar concentrations of loratadine, astemizole and ebastine sensitized NSCLC cells to chemotherapy and reverted multidrug resistance in NSCLC, breast and prostate cancer cells. Thus, CAD antihistamines may improve the efficacy of cancer chemotherapy.

Visualizing Lysosomal Membrane Permeabilization by Fluorescent Dextran Release.

Lysosomal membrane permeabilization (LMP) is an effective programmed cell death pathway triggered in response to a variety of cytotoxic stimuli and cellular conditions. In the method presented here, LMP is monitored by first taking advantage of the steady endocytic capacity of cells to load fluorescent dextran into lysosomes, and then simply observing the translocation of lysosomally localized dextran into the cytosol after an LMP-inducing insult. Fluorescent dextran in healthy cells appears in punctate structures representing intact lysosomes, whereas after LMP, a diffuse staining pattern throughout the cytoplasm is observed. Using this method, LMP can be followed in real time using time-lapse imaging. The size of pores formed in the membrane during LMP by size exclusion can also be determined using dextrans of different sizes and colors.