MSR1 repeats modulate gene expression and affect risk of breast and prostate cancer.

Background: MSR1 repeats are a 36-38 bp minisatellite element that have recently been implicated in the regulation of gene expression, through copy number variation (CNV). Patients and methods: Bioinformatic and experimental methods were used to assess the distribution of MSR1 across the genome, evaluate the regulatory potential of such elements and explore the role of MSR1 elements in cancer, particularly non-familial breast cancer and prostate cancer. Results: MSR1s are predominately located at chromosome 19 and are functionally enriched in regulatory regions of the genome, particularly regions implicated in short-range regulatory activities (H3K27ac, H3K4me1 and H3K4me3). MSR1-regulated genes were found to have specific molecular roles, such as serine-protease activity (P = 4.80 × 10-7) and ion channel activity (P = 2.7 × 10-4). The kallikrein locus was found to contain a large number of MSR1 clusters, and at least six of these showed CNV. An MSR1 cluster was identified within KLK14, with 9 and 11 copies being normal variants. A significant association with the 9-copy allele and non-familial breast cancer was found in two independent populations (P = 0.004; P = 0.03). In the white British population, the minor allele conferred an increased risk of 1.21-3.51 times for all non-familial disease, or 1.7-5.3 times in early-onset disease. The 9-copy allele was also found to be associated with increased risk of prostate cancer in an independent population (odds ratio = 1.27-1.56; P =0.009). Conclusions: MSR1 repeats act as molecular switches that modulate gene expression. It is likely that CNV of MSR1 will affect risk of development of various forms of cancer, including that of breast and prostate. The MSR1 cluster at KLK14 represents the strongest risk factor identified to date in non-familial breast cancer and a significant risk factor for prostate cancer. Analysis of MSR1 genotype will allow development of precise stratification of disease risk and provide a novel target for therapeutic agents.

Cancer treatment-related neuropathic pain: proof of concept study with menthol--a TRPM8 agonist.

PURPOSE: Effective treatment of neuropathic pain without unacceptable side effects is challenging. Cancer sufferers increasingly live with long-term treatment-related neuropathic pain, resulting from chemotherapy-induced peripheral neuropathy (CIPN) or surgical scars. This proof-of-concept study aimed to determine whether preclinical evidence for TRPM8 ion channels in sensory neurons as a novel analgesic target could be translated to clinical benefit in patients with neuropathic pain, using the TRPM8 activator menthol. PATIENTS AND METHODS: Patients with problematic treatment-related neuropathic pain underwent a baseline assessment using validated questionnaires, psychophysical testing, and objective functional measures. The painful area was treated with topical 1 % menthol cream twice daily. Assessments were repeated at 4-6 weeks. The primary outcome was the change in Brief Pain Inventory total scores at 4-6 weeks. Secondary outcomes included changes in function, mood and skin sensation. RESULTS: Fifty-one patients (female/male, 32/19) were recruited with a median age of 61 (ranging from 20 to 89). The commonest aetiology was CIPN (35/51), followed by scar pain (10/51). Thirty-eight were evaluable on the primary outcome. Eighty-two per cent (31/38) had an improvement in total Brief Pain Inventory scores (median, 47 (interquartile range, 30 to 64) to 34 (6 to 59), P < 0.001). Improvements in mood (P = 0.0004), catastrophising (P = 0.001), walking ability (P = 0.008) and sensation (P < 0.01) were also observed. CONCLUSION: This proof-of-concept study indicates that topical menthol has potential as a novel analgesic therapy for cancer treatment-related neuropathic pain. Improvements in patient-rated measures are supported by changes in objective measures of physical function and sensation. Further systematic evaluation of efficacy is required.

Flash glucose monitoring with the FreeStyle Libre 2 compared with self-monitoring of blood glucose in suboptimally controlled type 1 diabetes: the FLASH-UK randomised controlled trial protocol.

INTRODUCTION: Optimising glycaemic control in type 1 diabetes (T1D) remains challenging. Flash glucose monitoring with FreeStyle Libre 2 (FSL2) is a novel alternative to the current standard of care self-monitoring of blood glucose (SMBG). No randomised controlled trials to date have explored the potential benefits of FSL2 in T1D. We aim to assess the impact of FSL2 in people with suboptimal glycaemic control T1D in comparison with SMBG. METHODS: This open-label, multicentre, randomised (via stochastic minimisation), parallel design study conducted at eight UK secondary and primary care centres will aim to recruit 180 people age ≥16 years with T1D for >1 year and glycated haemoglobin (HbA1c) 7.5%-11%. Eligible participants will be randomised to 24 weeks of FSL2 (intervention) or SMBG (control) periods, after 2-week of blinded sensor wear. Participants will be assessed virtually or in-person owing to the COVID-19 pandemic. HbA1c will be measured at baseline, 12 and 24 weeks (primary outcome). Participants will be contacted at 4 and 12 weeks for glucose optimisation. Control participants will wear a blinded sensor during the last 2 weeks. Psychosocial outcomes will be measured at baseline and 24 weeks. Secondary outcomes include sensor-based metrics, insulin doses, adverse events and self-report psychosocial measures. Utility, acceptability, expectations and experience of using FSL2 will be explored. Data on health service resource utilisation will be collected. ANALYSIS: Efficacy analyses will follow intention-to-treat principle. Outcomes will be analysed using analysis of covariance, adjusted for the baseline value of the corresponding outcome, minimisation factors and other known prognostic factors. Both within-trial and life-time economic evaluations, informed by modelling from the perspective of the National Health Service setting, will be performed. ETHICS: The study was approved by Greater Manchester West Research Ethics Committee (reference 19/NW/0081). Informed consent will be sought from all participants. TRIAL REGISTRATION NUMBER: NCT03815006. PROTOCOL VERSION: 4.0 dated 29 June 2020.

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L.

BACKGROUND: Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies. METHODS: The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide. RESULTS: Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 microg/ml for yeasts, 125 to 500 microg/ml for Aspergillus species, and 7.81 to 62.5 microg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 x MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 x MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 x to 8 x MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol. CONCLUSIONS: The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.

Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining.

A rapid method for the flow microfluorometric determination of the DNA content per cell is described. Incubation of cells in a hypotonic solution of propidium iodide results in disruption of the cell membrane and rapid staining of nuclear chromatin. DNA distribution histograms generated from cells stained by this method are identical to those generated after fixation and RNase digestion. In contrast to some earlier described methods, the present technique is rapid (5 min of processing), requires a minimal amount of material, and avoids formation of cell clumps.

Cytochalasin-B: time-lapse cinematographic studies on its effects on cytokinesis.

L cells exposed to cytochalasin-B (cyto-B) show the normal development of deep cleavage furrows in both bipolar and multipolar cell divisions Due to the drug-induced inhibition of cellular motility, the resulting daughter cells do not move away from each other but reunite to form multinucleate cells. In mitotic cells from cultures exposed to cyto-B for long periods of time, vigorous blebbing and contraction of the cell surface is seen The evidence from time-lapse studies presented suggests that cyto-B-induced multinucleate cells are formed, not by the failure of the cleavage furrow, but by the drug-induced changes in surface activity and motility of cells after division.

Observations on the association of helical polyribosomes and filaments with vincristine-induced crystals in Earle's L-cell fibroblasts.

Earle's L-929 fibroblasts from cultures treated with 5-10 microg/ml of vincristine sulfate have a large number of eosinophilic, proteinaceous crystals in their cytoplasm. In electron micrographs, large arrays of helical polyribosomes, stacks of Golgi lamellae, and membranes of granular endoplasmic reticulum are seen in the cytoplasm of these cells. "Stalks" of fine granular material, approximately 300 A wide, are often seen in association with the arrays of the helical polyribosomes. In many sections rows of helical polyribosomes and filaments emerging from individual polyribosomes are seen in intimate contact with the crystals. A gradual reduction in the number of crystals and crystal-bearing cells is seen in cultures removed from the drug-containing medium and reincubated in fresh medium. In electron micrographs, these reincubated cells show large aggregates of filamentous material in the cytoplasm, and in many sections filaments are seen in continuity with the crystals.

Cytofluorometric studies on the action of podophyllotoxin and epipodophyllotoxins (VM-26, VP-16-213) on the cell cycle traverse of human lymphoblasts.

Flow microfluorometric analysis of human lymphoid cells exposed in vitro to cytostatic concentrations of podophyllotoxin (0.01-5 mug/ml for 24 h) shows that a major part of this population (40-60%) has the DNA content of cells in the G2-M part of the cell cycle, and that approximately 60% of these cells are arrested in mitosis. Although a similar pattern of DNA distribution is seen in cultures exposed to cytostatic concentrations of VM-26(0.01 mug/ml) and VP--16-213(0.1 mug/ml), no mitotic cells are seen in these cultures. Exposure to higher concentrations: of VM-26 (0.1 mug/ml) and VP-16-213 (1.0 mug/ml) inhibits cell cycle traverse, and after 24 hr of exposure a major part of the population is arrested with the DNA content of cell in the S part of the cell cycle. Exposure to higher drug concentrations leads to a reduction in the number of cells with the late S-G2DNA content. Whereas the cell cycle block induced by cytostatic concentrations of podophyllotoxin (0.01 mug/ml) is readily reversible by reincubation of cells in drug-free medium, cells blocked by VM-26 and VP-16-213 are unable to resume cell-cycle traverse under similar conditions.

Cell surface tubulin in leukemic cells: molecular structure, surface binding, turnover, cell cycle expression, and origin.

We report here new characteristics of cell surface tubulin from a human leukemia cell line. These cells (CEM cells) possess tubulin that is readily iodinated on the surface of living cells, turns over at a rate identical to that of other surface proteins, and is present throughout the cell cycle. When removed with trypsin, it rapidly returns to the surface. Peptide mapping of iodinated surface tubulin indicates that it possesses a similar, but not identical, primary structure to total CEM and rat brain tubulin. Living CEM cells are able to bind specifically a subfraction of CEM tubulin from metabolically labeled high speed supernatants of lysed CEM cells. Surface tubulin is more basic than the total tubulin pool. The binding, which is saturable, is inhibited by unlabeled CEM high speed supernatants but not by excess thrice-cycled rat or bovine brain tubulin. Surface tubulin is also shown to bind to living nontransformed normal rat kidney cells but not to normal, circulating, mononuclear white cells. Activated lymphocytes produce a tubulin that binds to CEM cells. Since CEM tubulin was detected in the media of 6-h cultures of CEM cells, we must conclude that at least some of the surface tubulin comes from the media. We further conclude that these leukemic cells produce an unusual tubulin that may bind specifically to any membrane. The presence of iodinatable surface tubulin, however, appears to require both the production of a unique tubulin and the presence of a "receptor-like" surface binding component.

Laparoscopic inguinal hernia repair: a rare case of colonic mesh migration.

Inguinal hernia repair can be performed via either an open or laparoscopic technique. Use of a mesh to repair the abdominal wall defect is now common practice, leading to a reduction in hernia recurrence but also associated with a number of complications. We report a rare case of a 49-year old man who presented 3 years after laparoscopic hernia repair with right-sided abdominal pain and loose stools. Colonoscopy and computed tomography revealed a mesh and fixation devices within the lumen of the caecum and ascending colon. The mesh was successfully excised with primary closure of the bowel defect. This case highlights the importance of recognising mesh migration as a complication of hernia repair, a phenomenon which can lead to serious morbidity. We suggest that patients should be informed of this risk during the consent process, while further research is needed to investigate how this occurrence can be prevented.

A standardized root extract of Withania somnifera and its major constituent withanolide-A elicit humoral and cell-mediated immune responses by up regulation of Th1-dominant polarization in BALB/c mice.

The effects of graded doses of a chemically standardized aqueous alcoholic (1:1) root extract (AGB) of Withania somnifera on the immune system of SRBC immunized BALB/c mice were investigated. Mice were administrated AGB orally for 15 days. AGB stimulated cell mediated immunity, IgM and IgG titers reaching peak value with 30 mg/kg b.wt. Flow cytometric analysis of lymphocyte surface markers of T cells (CD3(+), CD4(+) and CD8(+)) and B cells (CD19(+)) indicated prominent enhancement in proliferation and differentiation of lymphocytes. The extract selectively, induced type 1 immunity because it guided enhanced expression of T helper cells (Th)1 cytokines interferon (IFN)-gamma and interleukin (IL)-2 while Th2 cytokine IL-4 observed a moderate decline. Confirmation of Th1 polarization was obtained from augmented levels of IgG2a over IgG1 in the blood sera of AGB treated groups. Withanolide-A, a major constituent of AGB appeared responsible for Th1 skewing effect of the extract as it significantly increased the levels of Th1 cytokines, decreased moderately IL-4 and significantly restored the selective dexamethasone inhibition of Th1 cytokines in mouse splenocytes cultures in vitro. In addition, AGB also strongly activated macrophage functions ex vivo and in vitro indicated by enhanced secretion of nitrite, IL-12 and TNF-alpha. In contrast IL-10 remained unchanged again suggesting that AGB critically influenced Th1 profile of the cytokines. The studies suggested that AGB supports predominantly Th1 immunity with increase in macrophage functions. The standardized root extract of no toxicological consequences might therefore, find useful applications against the intracellular pathogens and in the management of immune suppressed diseases.

CYP17, SRD5A2, CYP1B1, and CYP2D6 gene polymorphisms with prostate cancer risk in North Indian population.

To investigate the involvement of the CYP17, SRD5A2, CYP1B1, and CYP2D6 variants with prostate cancer, a case-control study of 100 patients and an equal number of age-matched control men was conducted. There appears to be a nonsignificant increase with risk of prostate cancer for individuals carrying one copy of the CYP17 A2 allele (OR, 1.80; 95% CI, 0.99-3.29, P=0.05). The risk was increased in individuals having two A2 alleles (OR; 2.81, 95% CI, 1.06-7.40, P=0.03). Compared with men having the VV genotype of SRD5A2 gene, there was no significant association between the VL genotype and the risk of prostate cancer (OR; 0.54, 95% CI; 0.29-1.03, P=0.06). There was no difference in the occurrence of the genotype LL between controls and prostate cancer patients (OR; 0.90, 95% CI; 0.43-1.89, P=0.79). There was a nonsignificant increased risk of prostate cancer for individuals carrying the CYP1B1Leu/Val genotype (OR, 1.70, 95% CI, 0.91-3.17, P =0.09), which was increased in those having the Val/Val allele (OR, 3.38; 95% CI, 1.13-10.07, P=0.02). Relative to men homozygous for the wild-type allele in CYP2D6 gene, those heterozygous for the B allele had an odds ratio of 1.78 (95% CI, 0.76-4.17, P=0.18) for patients, and for homozygous individuals, it was 1.95 (0.55-6.93, P=0.30). These observations have suggested that the CYP17 A2/A2, CYP1B1 Val/Val, and CYP2D6 genotypes may be associated with an altered risk of prostate cancer, while the CYP2D6 and SRD5A2 V89L polymorphism have no association with its risk in the North Indian population.

Phytochemical and genetic analysis in selected chemotypes of Withania somnifera.

The main active components and genetic profile of 15 selected accessions of Withania somnifera Dunal. were analysed. Ethanolic extract of the dried roots/leaves of the plant was concentrated under pressure at 50+/-5 degrees C and was analysed for main compounds (withanolides and withaferin A) by HPLC. All the main components were found to be present in accessions (AGB 002, AGB 009, RSS 009, RSS 033). Correlation of these main components with their genetic factors, was undertaken using AFLP (amplified fragment length polymorphism) markers. Among 64 primers 7 yielded optimum polymorphism. A total of 913 polymorphic peaks were generated using these primers. Jaccard's similarity coefficient indicated that accessions having almost the same active compounds clustered together. The present study demonstrates that AFLP can be successfully used to resolve the correlation of AFLP data with the presence of secondary metabolites.

Intracellular adriamycin levels and cytotoxicity in adriamycin-sensitive and adriamycin-resistant P388 mouse leukemia cells.

Adriamycin (ADR) (NSC-123127) uptake and retention in ADR-sensitive P388 leukemia (P388/S) and ADR-resistant P388 leukemia (p388/R) cells were compared by fluorometry and laser flow cytometry (FCM) and were correlated with cytotoxic effects. Drug levels in P388/R cells treated in vitro with ADR (1-10 micrograms/ml) were twofold to fourfold lower than were levels in similarly treated P388/S cells FCM analysis of P388/S and P388/R cells exposed in vitro to ADR showed qualitative and quantitative differences in ADR fluorescence profiles of drug-treated cells (1-5 micrograms/ml) but not of the isolated nuclei (0.5- 10 micrograms/ml). Drug-induced perturbations in cell cycle traverse and chromosome aberrations were seen in P388/S but not in P388/R cells treated with 0.5-5 micrograms ADR/ml in vitro or 4-8 mg ADR/kg in vivo. The role of FCM in rapidly comparing and quantitating cellular ADR fluorescence profiles of ADR-sensitive and ADR-resistant tumors was demonstrated.

Laser flow cytometric studies on the intracellular fluorescence of anthracyclines.

We have used a laser flow cytometer to excite and quantitate the intracellular fluorescence of cells exposed in vitro and in vivo to various anthracyclines. In cells exposed to Adriamycin (ADR), intracellular drug fluorescence appeared slowly and reached a peak after 4 hr of incubation. Cells incubated with 10 micrograms/ml were 5 times more fluorescent than were cells incubated with 1 microgram/ml. Cells exposed to daunomycin were 2 to 4 times more fluorescent than were cells similarly exposed to ADR, and the intracellular appearance of daunomycin fluorescence was much more rapid. Cells exposed to N-trifluoroacetyladriamycin and carminomycin had higher amounts of intracellular fluorescence (2 to 4 times), and peak values were reached much more rapidly than in cells exposed to ADR. In cells exposed to rubidazone, fluorescence increased 2- to 4-fold with increased drug concentration and length of exposure. In contrast, nogalamycin fluorescence reached a peak after 60 min of incubation, and a 10-fold increase in drug concentration increased fluorescence only 2-fold. In animals given injections of ADR (4 mg/kg) and sacrificed after 3 hr, drug fluorescence could be detected in tumor and spleen cells. In contrast, fluorescence in heart nuclei was barely recognizable. However, incubation of isolated nuclei in ADR (1 microgram/ml) showed that bone marrow and heart nuclei had greater amounts of ADR fluorescence (2- to 3-fold) than did spleen or liver nuclei similarly treated. The use of laser flow cytometry for monitoring intracellular anthracycline transport, binding, and efflux is demonstrated.

Morphological basis for the cytolytic effect of vinblastine and vincristine on cultured human leukemic lymphoblasts.

Exposure of human leukemic lymphoblasts in suspension cultures to low concentrations of vinblastine and vincristine results in alterations in cell shape and leads to the formation and release of a large number of membrane-lined vesicles from the cytoplasm. Separation of these vesicles from peripheral cytoplasm is effected through alignment and fusion of small vacoules. Similar vesicle formation is seen neither in fibroblasts exposed to vinblastine nor in lymphoblasts exposed to bleomycin or adriamycin. Possible relation of this phenomenon to vinblastine- and vincristine-induced cytotoxicity, spherocytosis, and thrombocytosis is discussed.

Effect of oxygen concentration on the growth and drug sensitivity of human melanoma cells in soft-agar clonogenic assay.

Tumor cells from a human melanoma xenograft show better colony growth and plating efficiency in soft agar when incubated in a 5% O2 atmosphere that at a higher concentration of 20% O2. Exposure to low drug concentrations under a 20% (but not 5%) O2 atmosphere stimulated plating efficiency. Enhanced sensitivity (4- to 20-fold) to a variety of chemotherapeutic agents was seen in cells incubated under low but not the higher O2 concentration. These studies suggest the important role of O2 concentration in tumor colony growth, plating efficiency, and drug cytotoxicity for human solid tumors in vitro.

Effect of 2,3-dihydro-1 H-imidazo[1,2-b]pyrazole on the proliferation of mouse leukemic and normal cells in vivo.

Administration of 2,3-dihydro-1 H-imidazo[1,2-b]pyrazole (IMPY; NSC 51143), 250 to 500 mg/kg, in Day 5 L1210 and P388 (ascites) tumor-bearing mice did not consistently prolong the life span of tumor-bearing animals. Flow cytometry and autoradiographic studies showed that, after 12 to 18 hr of a single IMPY injection, both P388 and L1210 tumor cells were synchronized in S phase. In contrast, IMPY inhibited cellular proliferation in both bone marrow and duodenal crypts during the first 24 hr. and a recovery was detectable only after 36 hr, returning to pretherapy values by 72 hr. Preliminary data indicate that this differential response of normal versus tumor cells to IMPY can be exploited to maximize chemotherapeutic efficacy in scheduled chemotherapy with cycle-specific agents.

Effect of adriamycin on the cell cycle traverse and kinetics of cultured human lymphoblasts.

Exposure of cultured human lymphoblasts to adriamycin (ADM) (0.1 mug/ml for 24 hr or 0.5 mug/ml for 1 hr) leads to an accumulation of cells with the DNA content of late S and G2. Higher concentrations of ADM (0.5 to 10 mug/ml) inhibit cell cycle traverse. Effect of ADM on cell cycle traverse, cell growth, and incorporation of labeled precursors into DNA is dependent on drug concentration and length of exposure to ADM. Synchronized cells in G1 or G2 part of the cell cycle are less sensitive to ADM than cells in S phase. Similarly, plateau-phase cells are less sensitive to ADM than cells from log-phase cultures.

Thaliblastine, a plant alkaloid, circumvents multidrug resistance by direct binding to P-glycoprotein.

The effect of thaliblastine (TBL, NSC-68075), a plant alkaloid, in over-coming multidrug resistance was investigated in doxorubicin (ADR)-resistant murine leukemic P388/R-84 cells. In the soft agar clonogenic assay, a nontoxic concentration of TBL (2 microM) reduced the 50% inhibitory dose of ADR (1-h exposure) from 10.8 to 1.4 microM with a dose modification factor of 7.7. Continuous treatment of P388/R-84 cells with ADR and TBL for 24 h further lowered the 50% inhibitory dose from 3.5 to 0.07 microM, the resistance level being decreased from 233-fold in the absence of TBL to 4.7-fold in the presence of TBL as compared to the parental P388 cells. Although ADR or TBL individually had no detectable effects on cell cycle traverse, the combination of the two drugs caused a significant G2 block. Flow cytometric analysis showed that TBL enhanced ADR retention in P388/R-84 cells in a dose- and time-dependent manner. TBL partially blocked the photolabeling of P-glycoprotein with [3H]azidopine, and this blocking effect was further enhanced in combination with ADR. Our results indicate that TBL can reverse multidrug resistance by direct interaction with P-glycoprotein, thereby increasing cellular ADR retention.