Olive juice dry extract containing hydroxytyrosol, as a nontoxic and safe substance: Results from pre-clinical studies and review of toxicological studies.

Products derived from olives, such as the raw fruit and oils, are widely consumed due to their taste, and purported nutritional/health benefits. Phenolic compounds, especially hydroxytyrosol (HT), have been proposed as one of the key substances involved in these effects. An olive juice extract, standardized to contain 20% HT ("OE20HT"), was produced to investigate its health benefits. The aim of this study was to demonstrate the genotoxic safety of this ingredient based on in vitro Ames assay and in vitro micronucleus assay. Results indicated that OE20HT was not mutagenic at concentrations of up to 5000 µg/plate, with or without metabolic activation, and was neither aneugenic nor clastogenic after 3-hour exposure at concentrations of up to 60 µg/mL with or without metabolic activation, or after 24-hour exposure at concentrations of up to 40 µg/mL. To further substantiate the safety of OE20HT following ingestion without conducting additional animal studies, a comprehensive literature review was conducted. No safety concerns were identified based on acute or sub-chronic studies in animals, including reproductive and developmental studies. These results were supported by clinical studies demonstrating the absence of adverse effects after oral supplementation with olive extracts or HT. Based on in vitro data and the literature review, the OE20HT extract is therefore considered as safe for human consumption at doses up to 2.5 mg/kg body weight/day.

Updates on the Diagnosis and Management of Glaucoma.

Glaucoma is the leading cause of blindness throughout the world (after cataracts); therefore, general physicians should be familiar with the diagnosis and management of affected patients. Glaucomas are usually categorized by the anatomy of the anterior chamber angle (open vs narrow/closed), rapidity of onset (acute vs chronic), and major etiology (primary vs secondary). Most glaucomas are primary (ie, without a contributing comorbidity); however, several coexisting ophthalmic conditions may serve as the underlying etiologies of secondary glaucomas. Chronic glaucoma occurs most commonly; thus, regular eye examinations should be performed in at-risk patients to prevent the insidious loss of vision that can develop before diagnosis. Glaucoma damages the optic nerve and retinal nerve fiber layer, leading to peripheral and central visual field defects. Elevated intraocular pressure (IOP), a crucial determinant of disease progression, remains the only modifiable risk factor; thus, all current treatments (medications, lasers, and operations) aim to reduce the IOP. Pharmacotherapy is the usual first-line therapy, but noncompliance, undesirable adverse effects, and cost limit effectiveness. Laser and surgical treatments may lower IOP significantly over long periods and may be more cost effective than pharmacotherapy, but they are plagued by greater procedural risks and frequent treatment failures. Traditional incisional procedures have recently been replaced by several novel, minimally invasive glaucoma surgeries with improved safety profiles and only minimal decreases in efficacy. Minimally invasive glaucoma surgeries have dramatically transformed the surgical management of glaucoma; nevertheless, large, randomized trials are required to assess their long-term efficacy.

Intensity modulated radiotherapy for anal canal squamous cell carcinoma: A 16-year single institution experience.

INTRODUCTION: To report long-term efficacy and adverse events (AEs) associated with intensity modulated radiotherapy (IMRT) for patients with anal canal squamous cell carcinoma (ASCC). MATERIALS AND METHODS: This was a retrospective review of patients with ASCC who received curative-intent IMRT and concurrent chemotherapy (98%) between 2003 and 2019. Overall survival (OS), colostomy-free survival (CFS), and progression-free survival (PFS) were estimated using the Kaplan-Meier method. The cumulative incidence of local recurrence (LR), locoregional recurrence (LRR), and distant metastasis (DM) were reported. Acute and late AEs were recorded per National Cancer Institute Common Terminology Criteria for AEs. RESULTS: 127 patients were included. The median patient age was 63 years (interquartile range [IQR] 55-69) and 79% of patients were female. 33% of patients had T3-4 disease and 68% had clinically involved pelvic or inguinal lymph nodes (LNs).The median patient follow-up was 47 months (IQR: 28-89 months). The estimated 4-year OS, CFS, and PFS were 81% (95% confidence interval [CI]: 73%-89%), 77% (95% CI: 68%-86%), and 78% (95% CI: 70%-86%), respectively. The 4-year cumulative incidences of LR, LRR, and DM were 3% (95% CI: 1%-9%), 9% (95% CI: 5%-17%), and 10% (95% CI: 6%-18%), respectively. Overall treatment duration greater than 39 days was associated with an increased risk of LRR (Hazard Ratio [HR]: 5.2, 95% CI: 1.4-19.5, p = 0.015). The most common grade 3+ acute AEs included hematologic (31%), gastrointestinal (GI) (17%), dermatologic (16%), and pain (15%). Grade 3+ late AEs included: GI (3%), genitourinary (GU) (2%), and pain (1%). Current smokers were more likely to experience grade 3+ acute dermatologic toxicity compared to former or never smokers (34% vs. 7%, p < 0.001). CONCLUSIONS: IMRT was associated with favorable toxicity rates and long-term efficacy. These data support the continued utilization of IMRT as the preferred treatment technique for patients with ASCC.

Primary recurrent orbital schwannoma treated with surgical excision and Mitomycin-C.

PURPOSE: Primary orbital schwannoma (POS) is a slow growing, benign encapsulated peripheral nerve sheath tumor that occurs infrequently within the orbit. Recurrence of POS is extremely rare. Previous speculations for reasons of recurrence include incomplete excision and tumor seeding. OBSERVATIONS: We present the fifth case reported in the literature to date of POS that had 2 episodes of recurrences within 8 years after diagnosis, in which rapid and insidious relapses were observed after initial surgical resection. This is also the first reported recurrent POS in which topical Mitomycin-C (MMC) has been employed during surgical excision with an aim to prevent further recurrences. CONCLUSIONS: AND IMPORTANCE: Whilst complete surgical excision remained the standard for management for most POS, when there are multiple recurrences and in cases where total excision is not possible, addition of topical MMC may be an option that may bring about tumour regression as demonstrated in our case.

RADIANCE - Radiochemotherapy with or without Durvalumab in the treatment of anal squamous cell carcinoma: A randomized multicenter phase II trial.

PURPOSE: Anal squamous cell carcinomas (ASCC) are increasing in frequency across the developed world. The 3-year disease-free survival (DFS) in patients with locally-advanced disease is approximately 60% after primary radiochemotherapy (RCT). There is a strong rationale for combining immunotherapy with RCT in patients with ASCC due to its association with human papilloma virus (HPV) infection. METHODS/DESIGN: RADIANCE is an investigator initiated, prospective, multicenter, randomized phase II trial testing the addition of Durvalumab, a PD-L1 immune checkpoint inhibitor, to standard RCT in 178 patients with locally advanced ASCC (T2 ≥ 4 cm Nany, cT3-4 and/or cN+). In the control arm, patients will be treated with standard mitomycin C (MMC)/5-fluorouracil (5-FU)-based RCT. Intensity-modulated radiotherapy (IMRT) will be applied as follows: PTV_A (primary tumor) T1-T2 < 4 cm N+: 28 × 1.9 Gy = 53.2 Gy; or T2 ≥ 4 cm, T3-4 Nany: 31 × 1.9 Gy = 58.9 Gy; PTV_N (involved node): 28 × 1.8 Gy = 50.4 Gy ; and PTV_Elec (elective node): 28 × 1.43 Gy = 40.0 Gy over a period of 5,5-6 weeks. Concomitant chemotherapy will be administered using MMC with 5-FU during weeks 1 and 5 of radiotherapy (MMC 12 mg/m2, day 1 [maximum single dose 20 mg]; 5-FU 1000 mg/m2 days 1-4 and 29-32). In the experimental arm, Durvalmab (1500 mg absolute dose, intravenously) will be combined with the same RCT as in the control arm. Immunotherapy with Durvalumab will start 14 days before initiation of standard RCT, administered every four weeks (q4w) thereafter for a total of twelve doses. The primary endpoint is disease-free survival (DFS) after 3 years. DISCUSSION: As ASCC is considered an immunogenically "hot" tumor due to its association with HPV infection, the combination of RCT with Durvalumab may improve tumor control and long-term clinical outcome in this patient collective compared to RCT alone.

Cancer Therapy-Related Cardiac Dysfunction of Nonanthracycline Chemotherapeutics: What Is the Evidence?

Cancer therapy-related cardiac dysfunction (CTRCD) is one of the most concerning cardiovascular side effects of cancer treatment. Important reviews within the field of cardio-oncology have described various agents to be associated with a high risk of CTRCD, including mitomycin C, ifosfamide, vincristine, cyclophosphamide, and clofarabine. The aim of this study was to provide insight into the data on which these incidence rates are based. We observed that the reported cardiotoxicity of mitomycin C and ifosfamide is based on studies in which most patients received anthracyclines, complicating the interpretation of their association with CTRCD. The high incidence of vincristine-induced cardiotoxicity is based on an incorrect interpretation of a single study. Incidence rates of clofarabine remain uncertain due to a lack of cardiac screening in clinical trials. The administration of high-dose cyclophosphamide (>1.5 g/m2/day) is associated with a high incidence of CTRCD. Based on our findings, a critical re-evaluation of the cardiotoxicity of these agents is warranted.

Magnesium stearate, a widely-used food additive, exhibits a lack of in vitro and in vivo genotoxic potential.

Magnesium stearate is widely used in the production of dietary supplement and pharmaceutical tablets, capsules and powders as well as many food products, including a variety of confectionery, spices and baking ingredients. Although considered to have a safe toxicity profile, there is no available information regarding its potential to induce genetic toxicity. To aid safety assessment efforts, magnesium sulfate was evaluated in a battery of tests including a bacterial reverse mutation assay, an in vitro chromosome aberration assay, and an in vivo erythrocyte micronucleus assay. Magnesium stearate did not produce a positive response in any of the five bacterial strains tested, in the absence or presence of metabolic activation. Similarly, exposure to magnesium stearate did not lead to chromosomal aberrations in CHL/IU Chinese hamster lung fibroblasts, with or without metabolic activation, or induce micronuclei in the bone marrow of male CD-1 mice. These studies have been used by the Japanese government and the Joint FAO/WHO Expert Committee on Food Additives in their respective safety assessments of magnesium stearate. These data indicate a lack of genotoxic risk posed by magnesium stearate consumed at current estimated dietary exposures. However, health effects of cumulative exposure to magnesium via multiple sources present in food additives may be of concern and warrant further evaluation.

Genotoxicity evaluation of alpha-linolenic acid-diacylglycerol oil.

The alpha-linolenic acid (ALA)-diacylglycerol (DAG) oil is an edible oil enriched with DAG (>80%) and ALA (>50%). Although DAG oil, which mainly consists of oleic and linoleic acids has no genotoxic concerns, the fatty acid composition could affect the chemical property of DAG. Therefore, the purpose of this study was to evaluate the genotoxicity of ALA-DAG oil using standard genotoxicity tests in accordance with the OECD guidelines. ALA-DAG oil showed negative results in the bacterial reverse mutation test (Ames test) and in vitro micronucleus test in cultured Chinese hamster lung cells with and without metabolic activation, and in the in vivo bone marrow micronucleus test in mice. Our results did not show any genotoxicity, suggesting that the fatty acid composition had no deleterious effects. We conclude that ALA-DAG oil had no genotoxicity concerns under the testing conditions.

Human CST abundance determines recovery from diverse forms of DNA damage and replication stress.

Mammalian CST (CTC1-STN1-TEN1) is a telomere-associated complex that functions in telomere duplex replication and fill-in synthesis of the telomeric C-strand following telomerase action. CST also facilitates genome-wide replication recovery after HU-induced fork stalling by increasing origin firing. CTC1 and STN1 were originally isolated as a DNA polymerase α stimulatory factor. Here we explore how CST abundance affects recovery from drugs that cause different types of DNA damage and replication stress. We show that recovery from HU and aphidicolin induced replication stress is increased by CST over-expression. Elevated CST increases dNTP incorporation and origin firing after HU release and decreases the incidence of anaphase bridges and micronuclei after aphidicolin removal. While the frequency of origin firing after HU release is proportional to CST abundance, the number of cells entering S-phase to initiate replication is unchanged by CST overexpression or STN1 depletion. Instead the CST-related changes in origin firing take place in cells that were already in S-phase at the time of HU addition, indicating that CST modulates firing of late or dormant origins. CST abundance also influences cell viability after treatment with HU, aphidicolin, MMS and camptothecin. Viability is increased by elevated CST and decreased by STN1 depletion, indicating that endogenous CST levels are limiting. However, CST abundance does not affect viability after MMC treatment. Thus, CST facilitates recovery from many, but not all, forms of exogenous DNA damage. Overall our results suggest that CST is needed in stoichiometric amounts to facilitate re-initiation of DNA replication at repaired forks and/or dormant origins.

Protein kinase C ß inhibition by enzastaurin leads to mitotic missegregation and preferential cytotoxicity toward colorectal cancer cells with chromosomal instability (CIN).

Enzastaurin is a selective inhibitor of protein kinase C ß and a potent inhibitor of tumor angiogenesis. In addition, enzastaurin shows direct cytotoxic activity toward a subset of tumor cells including colorectal cancer cells (CRC). In spite of promising results in animal models, the clinical activity of enzastaurin in CRC patients has been disappointing although a subset of patients seems to derive benefit. In the present study we investigated the biological and cytotoxic activities of enzastaurin toward a panel of well-characterized CRC cell lines in order to clarify the mechanistic basis for the cytotoxic activity. Our results show that enzastaurin is significantly more cytotoxic toward CRC cells with chromosome instability (CIN) compared to cells with microsatellite instability (MSI). Since CIN is usually attributed to mitotic dysfunction, the influence of enzastaurin on cell cycle progression and mitotic transit was characterized for representative CIN and MSI cell lines. Enzastaurin exposure was accompanied by prolonged metaphase arrest in CIN cells followed by the appearance of tetraploid and micronuclei-containing cells as well as by increased apoptosis, whereas no detectable mitotic dysfunctions were observed in MSI cells exposed to isotoxic doses of enzastaurin. Our study identifies enzastaurin as a new, context dependent member of a heterogeneous group of anticancer compounds that induce "mitotic catastrophe," that is mitotic dysfunction accompanied by cell death. These data provide novel insight into the mechanism of action of enzastaurin and may allow the identification of biomarkers useful to identify CRC patients particularly likely, or not, to benefit from treatment with enzastaurin.

A knockdown with smoke model reveals FHIT as a repressor of Heme oxygenase 1.

Fragile histidine triad (FHIT) gene deletions are among the earliest and most frequent events in carcinogenesis, particularly in carcinogen-exposed tissues. Though FHIT has been established as an authentic tumor suppressor, the mechanism underlying tumor suppression remains opaque. Most experiments designed to clarify FHIT function have analyzed the consequence of re-expressing FHIT in FHIT-negative cells. However, carcinogenesis occurs in cells that transition from FHIT-positive to FHIT-negative. To better understand cancer development, we induced FHIT loss in human bronchial epithelial cells with RNA interference. Because FHIT is a demonstrated target of carcinogens in cigarette smoke, we combined FHIT silencing with cigarette smoke extract (CSE) exposure and measured gene expression consequences by RNA microarray. The data indicate that FHIT loss enhances the expression of a set of oxidative stress response genes after exposure to CSE, including the cytoprotective enzyme heme oxygenase 1 (HMOX1) at the RNA and protein levels. Data are consistent with a mechanism in which Fhit protein is required for accumulation of the transcriptional repressor of HMOX1, Bach1 protein. We posit that by allowing superinduction of oxidative stress response genes, loss of FHIT creates a survival advantage that promotes carcinogenesis.

BRCA1 and FancJ cooperatively promote interstrand crosslinker induced centrosome amplification through the activation of polo-like kinase 1.

DNA damage response (DDR) and the centrosome cycle are 2 of the most critical cellular processes affecting the genome stability in animal cells. Yet the cross-talks between DDR and the centrosome are poorly understood. Here we showed that deficiency of the breast cancer 1, early onset gene (BRCA1) induces centrosome amplification in non-stressed cells as previously reported while attenuating DNA damage-induced centrosome amplification (DDICA) in cells experiencing prolonged genotoxic stress. Mechanistically, the function of BRCA1 in promoting DDICA is through binding and recruiting polo-like kinase 1 (PLK1) to the centrosome. In a recent study, we showed that FancJ also suppresses centrosome amplification in non-stressed cells while promoting DDICA in both hydroxyurea and mitomycin C treated cells. FancJ is a key component of the BRCA1 B-complex. Here, we further demonstrated that, in coordination with BRCA1, FancJ promotes DDICA by recruiting both BRCA1 and PLK1 to the centrosome in the DNA damaged cells. Thus, we have uncovered a novel role of BRCA1 and FancJ in the regulation of DDICA. Dysregulation of DDR or centrosome cycle leads to aneuploidy, which is frequently seen in both solid and hematological cancers. BRCA1 and FancJ are known tumor suppressors and have well-recognized functions in DNA damage checkpoint and DNA repair. Together with our recent findings, we demonstrated here that BRCA1 and FancJ also play an important role in centrosome cycle especially in DDICA. DDICA is thought to be an alternative fail-safe mechanism to prevent cells experiencing severe DNA damage from becoming carcinogenic. Therefore, BRCA1 and FancJ are potential liaisons linking early DDR with the DDICA. We propose that together with their functions in DDR, the role of BRCA1 and FancJ in the activation of DDICA is also crucial for their tumor suppression functions in vivo.

The Fanconi anemia ID2 complex: dueling saxes at the crossroads.

Fanconi anemia (FA) is a rare recessive genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility in early adulthood. FA is caused by biallelic germ-line mutation of any one of 16 genes. While several functions for the FA proteins have been ascribed, the prevailing hypothesis is that the FA proteins function cooperatively in the FA-BRCA pathway to repair damaged DNA. A pivotal step in the activation of the FA-BRCA pathway is the monoubiquitination of the FANCD2 and FANCI proteins. Despite their importance for DNA repair, the domain structure, regulation, and function of FANCD2 and FANCI remain poorly understood. In this review, we provide an overview of our current understanding of FANCD2 and FANCI, with an emphasis on their posttranslational modification and common and unique functions.

Estrogenic and chemopreventive activities of xanthones and flavones of Syngonanthus (Eriocaulaceae).

The possible benefits of some bioactive flavones and xanthones present in plants of the genus Syngonanthus prompted us to screen them for estrogenic activity. However, scientific research has shown that such substances may have undesirable properties, such as mutagenicity, carcinogenicity and toxicity, which restrict their use as therapeutic agents. Hence, the aim of this study was to assess the estrogenicity and mutagenic and antimutagenic properties. We used recombinant yeast assay (RYA), with the strain BY4741 of Saccharomyces cerevisiae, and Ames test, with strains TA100, TA98, TA97a and TA102 of Salmonella typhimirium, to evaluate estrogenicity, mutagenicity and antimutagenicity of methanolic extracts of Syngonanthus dealbatus (S.d.), Syngonanthus macrolepsis (S.m.), Syngonanthus nitens (S.n.) and Syngonanthus suberosus (S.s.), and of 9 compounds isolated from them (1=luteolin, 2=mix of A-1,3,6-trihydroxy-2-methoxyxanthone and B-1,3,6-trihydroxy-2,5-dimethoxyxanthone, 3=1,5,7-trihydroxy-3,6-dimethoxyxanthone, 4=1,3,6,8-tetrahydroxy-2,5-dimethoxyxanthone, 5=1,3,6,8-tetrahydroxy-5-methoxyxanthone, 6=7-methoxyluteolin-8-C-ß-glucopyranoside, 7=7-methoxyluteolin-6-C-ß-glucopyranoside, 8=7,3'-dimethoxyluteolin-6-C-ß-glucopyranoside and 9=6-hydroxyluteolin). The results indicated the estrogenic potential of the S. nitens methanol extract and four of its isolated xanthones, which exhibited, respectively, 14.74±1.63 nM; 19.54±6.61; 7.20±0.37; 6.71±1.02 e 10.01±4.26 nM of estradiol-equivalents (EEQ). None of the extracts or isolated compounds showed mutagenicity in any of the test strains and all of them showed antimutagenic potential, in particular preventing mutations caused by aflatoxin B1 (AFB1) and benzo[a]pyrene (B[a]P). The results show that the xanthones, only isolated from the methanol extract of S. nitens capitula, probably were the responsible for its estrogenic activity and could be useful as phytoestrogens, providing a new opportunity to develop hormonal agents. In addition, flavones and xanthones could also be used as a new antimutagenic agent. Since, the mutagens are involved in the initiation and promotion of several human diseases, including cancer, the significance of novel bioactive phytocompounds in counteracting these pro-mutagenic and carcinogenic effects is now gaining credence.

The recurrence of pterygium after different modalities of surgical treatment.

PURPOSE: To assess and compare the recurrence rate of pterygium after limbal stem cell transplantation versus amniotic membrane transplantation (AMT) as ocular surface reconstructing measures in recurrent pterygium, also evaluation of the use of antimetabolite drugs as adjunctive therapy for AMT. PATIENTS AND METHODS: Prospective randomized comparative study included 60 eyes of 48 patients with recurrent pterygia. Informed consents were taken from all patients. Primary pterygium excision was performed 6-15 months ago; the cases were divided randomly into three equal groups: Group 1: included 20 eyes with excision of the pterygium and application of limbal stem cell transplantation with conjunctival autograft, group 2: included 20 eyes with excision of the pterygium followed by AMT and group 3: included 20 eyes in which surgical excision of pterygium was followed by intra-operative application of low-dose of MMC (0.05%) for 3 min then using AMT. RESULTS: The study included 36 males and 12 females of age ranged from 28 to 52 years. The recurrence rate was 2 eyes in group 1 (10%) (limbal stem cell transplantation + conjunctival autograft), 6 eyes in group 2 (30%) (AMT) and 4 eyes (20%) in group 3 (MMC + AMT). The rate of recurrence was significantly different between the three groups (P < 0.001). CONCLUSIONS: Limbal stem cell transplantation together with conjunctival autografting proved to be more effective in prevention of pterygium recurrence and in rapid restoration of normal epithelial morphology. MMC in addition to AMT decreases the incidence of recurrence.