Nuclear Morphometric Analysis Research Articles

Development and validation of betel-year: a clinical tool for quantifying exposure to betel quid chewing, utilizing nuclear morphometric analysis and validated by cytomorphological analysis

BackgroundBetel quid chewing, prevalent in Southeast Asia and South Asia, involves components such as betel leaf, areca nut, slaked lime, and sometimes tobacco. This study aims to assess buccal mucosa changes in betel quid chewers, develop a clinical tool for assessing exposure, and investigate its usability in predicting dysplasia.MethodsAfter obtaining ethical approval and informed consent, patients were recruited from the Out-Patient Department of Government Medical College, Omandurar, Government Estate, India. A target sample size of 200 was calculated. The data included the history of betel quid chewing, buccal mucosa cells obtained by oral cytology, and the severity of dysplasia of the slides assessed by pathologists. We utilized principal component analysis (PCA) and confirmatory factor analysis (CFA) to validate a new outcome variable reflecting nuclear morphometric parameters (NMPs). Multiplicative regression models were developed for betel years based on betel exposure and additives. Spearman correlation and Kruskal-Wallis test was used to check the association between betel years and dysplasia.ResultsSignificant differences in NMPs were observed among different betel chewing groups. We derived multiplicative regression models for betel years. In the logarithmic transformation approach, betel year = 0.05×betel-exposure×0.09×slaked-lime use×0.11×tobacco-use. In the original variable approach, betel year = 5.05×betel-exposure^0.00048×slaked-lime-use^0.18133×tobacco-use^1.47513. Spearman correlation and Kruskal–Wallis tests confirmed associations with dysplasia.ConclusionBetel year is a pioneering tool for assessing lifetime betel quid exposure, similar to pack years for smoking. It could aid in risk stratification, targeted interventions and shaping public health policies. Despite limitations, betel year holds promise for revolutionizing oral health risk assessment, and future research can expand its scope globally, considering diverse betel quid compositions.

In Vitro Assessment of the Cytotoxic Effect of 5-Fluorouracil, Thymoquinone and their Combination on Tongue Squamous Cell Carcinoma Cell Line.

Tongue cancer is the most prevalent type of oral cancer. Recently, natural compounds have been considered important resources for several anticancer drugs. Thymoquinone (TQ) exhibits a potent anti-cancer effect. 5-Fluorouracil (5-FU) is a chemotherapeutic drug that has been utilized in the treatment of cancer. Recently, combination therapy has gained popularity as a treatment option for patients with cancer. The present study was carried out to assess the cytotoxic effect of 5-Fluorouracil (5-FU), Thymoquinone (TQ), and their combination on tongue squamous cell carcinoma cell line (HNO-97). Tongue carcinoma cell line (HNO-97) was maintained in cultured flasks and the cells were divided into four groups; group Ι: control untreated group, group ΙΙ: HNO-97-treated cells with different concentrations of 5-FU from 0.5 µM/ml to 3µM/ml, group ΙIΙ: HNO-97-treated cells with different concentrations of TQ from 7.25µM/ml to 23.05µM/ml, and group ΙV: HNO-97-treated cells with both 5-FU and TQ in serial concentrations till (IC50) in a dose of 27.44 µM/ml. Determination of the cytotoxic effect of the tested agents on the HNO-97 cell line was done using methyl thiazole tetrazolium assay, nuclear morphometric analysis, microscopic examination, and annexin-v/ propidium iodide staining assay. The findings revealed that the cytotoxic effect of 5-FU, TQ, and their combination on tongue squamous cell carcinoma cell line (HNO-97) was dose-dependent. The microscopic examination revealed that 5-FU, TQ alone, or their combination induced apoptotic cell death. P-value < 0.05 was statistically significant. The combination of 5-FU and TQ produced a marked cytotoxic effect on HNO-97 cells.

Evaluation of nuclear morphometry in exfoliative cytology of buccal mucosa in patients with high risk of oral cancer

BackgroundOral cancer poses a significant global health burden, with India having the highest prevalence. Effective detection is crucial in effective prevention. This study aimed to evaluate nuclear morphometric parameters (NMPs) in buccal mucosa cells of smokers, correlate NMPs with dysplasia, establish cut off values for grading dysplasia, and investigate the relationship between NMPs and smoking. MethodsAfter obtaining ethical approval and informed consent, patients were recruited from the outpatient department of our institution. A target sample size of 250 was calculated. The data included smoking exposure quantified in pack-years, nuclear morphometric analysis (NMA) of buccal mucosa cells obtained through oral cytology using Image J, and the severity of dysplasia of the slides assessed by pathologists. Statistical analysis assessed the impact of dysplasia and the association between nuclear characteristics and smoking exposure. Receiver operating characteristic (ROC) plots determined the potential of these parameters to distinguish dysplasia levels. ResultsSignificant differences in NMPs were observed among different smoking groups. Dysplasia severity had a significant correlation with NMPs, and strong correlations were found between NMPs and lifetime smoking exposure. ROC analysis established cut off values for NMPs with good sensitivity and specificity for classifying dysplasia severity. ConclusionsThis study highlights the potential of NMA as a tool for oral cancer screening. NMPs can distinguish dysplasia severity and correlate with tobacco (smoking). The efficiency of NMA in a non-invasive oral cytology offers promise for patient-centered screening Additionally, the findings suggest future applications in telepathology and the potential for AI integration in automated screening after conducting multicentric large-scale studies.

Biodegradable nanocarrier of gemcitabine and tocopherol succinate synergistically ameliorates anti-proliferative response in MIA PaCa-2 cells

Gemcitabine (GEM) is an important chemotherapeutic agent used alone or in combination with other anticancer agents for the treatment of various solid tumors. In this study, the potential of a dietary supplement, α-tocopherol succinate (TOS) was investigated in combination with GEM by utilizing human serum albumin-based nanoparticles (HSA NPs). The developed nanoparticles were characterized using DLS, SEM and FTIR and evaluated in a panel of cell lines to inspect cytotoxic efficacy. The ratio metric selected combination of the NPs was further investigated in human pancreatic cancer cell line (MIA PaCa-2 cells) to assess the cellular death mechanism via a myriad of biochemical and bio-analytical assays including nuclear morphometric analysis by DAPI staining, ROS generation, MMP loss, intracellular calcium release, in vitro clonogenic assay, cell migration assay, cell cycle analysis, immunocytochemical staining followed by western blotting, Annexin V-FITC and cellular uptake studies. The desolvation-crosslinking method was used to prepare the NPs. The average size of TOS-HSA NPs and GEM-HSA NPs was found to be 189.47 ± 5 nm and 143.42 ± 7.4 nm, respectively. In combination, the developed nanoparticles exhibited synergism by enhancing cytotoxicity in a fixed molar ratio. The selected combination also significantly triggered ROS generation and mitochondrial destabilization, alleviated cell migration potential and clonogenic cell survival in MIA PaCa-2 cells. Further, cell cycle analysis, Annexin-V FITC assay and caspase-3 activation, up regulation of Bax and down regulation of Bcl-2 protein confirmed the occurrence of apoptotic event coupled with the G0/G1 phase arrest. Nanocarriers based this combination also offered approximately 14-folds dose reduction of GEM. Overall, the combined administration of TOS-HSA NPs and GEM-HSA NPs showed synergistic cytotoxicity accompanied with dose reduction of the gemcitabine. These encouraging findings could have implication in designing micronutrient based-combination therapy with gemcitabine and demands further investigation.

Ethyl Acetate Extract of Colletotrichum gloeosporioides Promotes Cytotoxicity and Apoptosis in Human Breast Cancer Cells.

Fungal endophytes are known to be a paragon for producing bioactive compounds with a variety of pharmacological importance. The current study aims to elucidate the molecular alterations induced by the bioactive compounds produced by the fungal endophyte Colletotrichum gloeosporioides in the tumor microenvironment of human breast cancer cells. GC/MS analysis of the ethyl acetate (EA) extract of C. gloeosporioides revealed the presence of bioactive compounds with anticancer activity. The EA extract of C. gloeosporioides exerted potential plasmid DNA protective activity against hydroxyl radicals of Fenton's reagent. The cytotoxic activity further revealed that MDA-MB-231 cells exhibit more sensitivity toward the EA extract of C. gloeosporioides as compared to MCF-7 cells, whereas non-toxic to non-cancerous HEK293T cells. Furthermore, the anticancer activity demonstrated by the EA extract of C. gloeosporioides was studied by assessing nuclear morphometric analysis and induction of apoptosis in MDA-MB-231 and MCF-7 cells. The EA extract of C. gloeosporioides causes the alteration in cellular and nuclear morphologies, chromatin condensation, long-term colony inhibition, and inhibition of cell migration and proliferation ability of MDA-MB-231 and MCF-7 cells. The study also revealed that the EA extract of C. gloeosporioides treated cells undergoes apoptosis by increased production of reactive oxygen species and significant deficit in mitochondrial membrane potential. Our study also showed that the EA extract of C. gloeosporioides causes upregulation of pro-apoptotic (BAX, PARP, CASPASE-8, and FADD), cell cycle arrest (P21), and tumor suppressor (P53) related genes. Additionally, the downregulation of antiapoptotic genes (BCL-2 and SURVIVIN) and increased Caspase-3 activity suggest the induction of apoptosis in the EA extract of C. gloeosporioides treated MDA-MB-231 and MCF-7 cells. Overall, our findings suggest that the bioactive compounds present in the EA extract of C. gloeosporioides promotes apoptosis by altering the genes related to the extrinsic as well as the intrinsic pathway. Further in vivo study in breast cancer models is required to validate the in vitro observations.

Evaluation of Nuclear Morphometry and Ki-67 Proliferative Marker in Astrocytomas: A Ambispective Study

Introduction: Astrocytomas are the commonest primary Central Nervous System (CNS) tumours. Its diagnosis is based on histopathological criteria defined by the World Health Organization (WHO) 2016 that grades astrocytoma into four grades. The subjective nature of WHO grading has prompted for more objective methods to evaluate nuclear features. Furthermore, Ki-67, a marker of cellular proliferation is a useful diagnostic tool that also helps in prognostic evaluation and to plan adjuvant therapy in astrocytomas. Aim: To evaluate the nuclear morphometry and Ki-67 proliferative marker in astrocytomas and to assess the relationship of WHO grade with proliferative activity using Ki-67 immunostaining. Materials and Methods: This ambispective study was conducted in the Department of Pathology, JSS Academy of Higher Education and Research, Mysuru, Karnataka, India, with a total duration of four years (October 2017 to September 2021). From 35 astrocytoma cases, Haematoxylin and Eosin (H&amp;E) stained slides were retrieved and reviewed appropriately by two pathologists and graded using the WHO criteria. Nuclear morphometric analysis was performed on the H&amp;E slides using Olympus BX-41 research microscope. The parameters evaluated were mean nuclear length, mean nuclear diameter, mean nuclear perimeter, mean nuclear area, Mean Nuclear Roundness Factor (MNRF) and mean nuclear ellipse form. The cases were then stained with Ki-67 antibody. The relationships between WHO grade of astrocytoma and nuclear morphometry and WHO grade with proliferative index was analysed. Results: According to WHO grading, four cases were Grade 1 (11.4%), 12 grade 2 (34.3%), six grade 3 (17.1%) and 13 grade 4 (37.1%). Significant correlation was seen between WHO grading and mean nuclear length, diameter, perimeter, area and Ki-67 with p-value of less than 0.001. Conclusion: Astrocytoma is an extremely heterogeneous disease with unpredictable outcome. The widely used WHO grading is subjective, while nuclear morphometry, using computer assisted image analysis, can ensure more objective assessment. The Ki-67 index could provide valuable information and may compliment the other parameters.

Retraction: Nuclear Morphometric Analysis of Leydig Cells of Male Pubertal Rats Exposed In Utero to Di(n-butyl) Phthalate.

[This retracts the article on p. 439 in vol. 26, PMID: 24526819.].

Comparison of Nonsequencing Techniques for Identification of NPM1 Mutations and Associated Blast Morphology in Patients With Acute Myeloid Leukemia.

Nucleophosmin 1 (NPM1) mutations affect 20% to 30% of all acute myeloid leukemia (AML) patients; several methods are employed to analyze NPM1 mutations, each of them with its advantages and limitations. To compare 3 nonsequencing protocols capable of detecting the main NPM1 mutations and to evaluate nuclear morphometric analysis (NMA) as an alternative to cuplike blast detection. We selected multiparameter flow cytometry (MFC), amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a quantitative PCR (qPCR) kit to identify NPM1 mutations in AML patients at diagnosis. We also evaluated the presence of cuplike blasts and assessed nuclear morphometry using NMA. MFC appears as a screening method for NPM1 mutations because of its lower specificity. ARMS-PCR demonstrated specificity similar to that of the qPCR kit, although it was more laborious. qPCR testing, conversely, is relatively fast and easy to standardize. Of these methods, qPCR was the only one capable of identifying the type of NPM1 mutation. With regard to morphology, NMA could be used as an alternative for the evaluation of cuplike blasts in AML smears. qPCR appears to be the best option to identify NPM1 mutations, with ARMS-PCR representing a cheaper alternative. MFC may be used as a screening method, in which results falling within and above the gray zone should be confirmed by molecular testing.

The Potential of Optimized Liposomes in Enhancement of Cytotoxicity and Apoptosis of Encapsulated Egyptian Propolis on Hep-2 Cell Line.

Purpose: Development of pharmaceutical dosage forms of natural products has gained great interest recently. Propolis is a natural product with various active compounds and multiple pharmacological activities. Its resinous nature and low bioavailability were obstacles in the optimum use of this magnificent natural product. Aim: This study evaluates the effect of using liposomes as a drug delivery system on the enhancement of the cytotoxic effect of propolis on squamous cell carcinoma cell lines (Hep-2) of head and neck. Methods: An optimized liposomal formulation of propolis was prepared using the conventional thin film hydration method 1, 2. The prepared (Hep-2) cell line was treated with different concentrations of propolis and optimized propolis liposomes for 24 h. The effect of both propolis and propolis liposomes on cell line was investigated using MTT assay, cytological examination, and nuclear morphometric analysis. The effect of the drugs on the cell apoptosis was evaluated using Annexin V. Results: The findings revealed that both propolis and propolis liposomes have a cytotoxic effect on Hep-2 cell line through induction of apoptosis. The effect was dose dependent. However, a statistically significant enhancement in propolis-mediated apoptosis on Hep-2 cells was elucidated due to encapsulation within the prepared liposomes. Conclusion: Liposome is a powerful tool for enhancing the cytotoxicity of propolis against Hep-2 cell line.

Morphological and mechanical changes induced by quercetin in human T24 bladder cancer cells

Quercetin is a flavonoid found in a great variety of foods such as vegetables and fruits. This compound has been shown to inhibit the proliferation of various types of cancer cells, as well as the growth of tumors in animal models. In the present study, we analyze morphological and mechanical changes produced by quercetin in T24 bladder cancer cells. Decreased cell viability and cell number were observed following quercetin treatment at 40 μM and 60 μM, respectively, as observed by the MTT assay and trypan blue exclusion test, supporting the hypothesis of quercetin anticancer effect. These assays also allowed us to determine the 40, 60, and 80 μM quercetin concentrations for the following analyses, Lactate Dehydrogenase assay (LDH); Nuclear Morphometric Analysis (NMA); and atomic force microscopy (AFM). The LDH assay showed no cytotoxic effect of quercetin on T24 cancer cells. The AFM showed morphological changes following quercetin treatment, namely decreased cell body, cytoplasmic retraction, and membrane condensation. Following quercetin treatment, the NMA evidenced an increased percentage of nuclei characteristic to the apoptotic and senescence processes. Cells also presented biophysical alterations consistent with cell death by apoptosis, as increased roughness and aggregation of membrane proteins, in a dose-dependent manner. Cellular elasticity, obtained through force curves, showed increased stiffness after quercetin treatment. Data presented herein demonstrate, for the first time, in a quantitative and qualitative form, the morphological and mechanical alterations induced by quercetin on bladder cancer cells.

Medulloblastoma: clinicopathological parameters, risk stratification, and survival analysis of immunohistochemically validated molecular subgroups

BackgroundMedulloblastoma (MB) is a heterogeneous disease, displaying distinct genetic profiles with specific molecular subgroups. This study aimed to validate MB molecular subgrouping using surrogate immunohistochemistry and associate molecular subgroups, histopathological types, and available clinicopathological parameters with overall survival (OS) and progression-free survival (PFS) of MB patients. This study included 40 MBs; immunohistochemical staining, using β-catenin and GRB2-Associated Binding Protein 1 (GAB1) antibodies, was used to classify MB cases into wingless signaling activated (WNT), sonic hedgehog (SHH), and non-WNT/SHH molecular subgroups. Nuclear morphometric analysis (for assessment of degree of anaplasia) and Kaplan-Meier survival curves were done.ResultsMB cases were classified into WNT (10%), SHH (30%), and non-WNT/SHH (60%) subgroups. Histopathological types differed significantly according to tumor location (p< 0.001), degree of anaplasia (p = 0.014), molecular subgroups (p < 0.001), and risk stratification (p = 0.008). Molecular subgroups differed significantly in age distribution (p = 0.031), tumor location (p< 0.001), histopathological variants (p < 0.001), and risk stratification (p < 0.001). OS was 77.5% and 50% after 1 and 2 years, while PFS was 65% and 27.5% after 1 and 2 years, respectively. OS and PFS were associated significantly with histopathological variants (p < 0.001 and 0.001), molecular subgroups (p = 0.012 and 0.005), and risk stratification (p < 0.001 and < 0.001), respectively.ConclusionsMedulloblastoma classification based on molecular subgroups, together with clinicopathological indicators, mainly histopathological types; accurately risk stratifies MB patients and predicts their survival.

Application of p16, p63, cyclin D1 immunostaining and nuclear morphometric analysis for assessment of cervical dysplasia

Background/Aim. Human papilloma virus (HPV) infection is the main etiological factor for the development of cervical precancerous dysplastic squamous intraepithelial lesions (SIL). The virus oncoproteins affect several proteins included in cell proliferation. The aim of this study was to evaluate application of immunohistochemical markers related to proteins of the cell cycle and, also, application of nuclear morphometric analysis for assessment of cervical dysplasia. Methods. Retrospective study included 78 women with detection of presence of high-risk HPV by polymerase chain reaction (PCR), with histopathology diagnosis low-grade SIL (LSIL) or high-grade SIL (HSIL). Immunohistochemical staining for p16, p63, cyclin D1 and morphometric analysis of the nuclear surface area were performed. The control group consisted of ten women without SIL and without HPV infection. This study was conducted in accordance with the Helsinki Declaration. Results. Comparing immunohistochemical expression of p16 and p63, highly statistically significant differences (p &lt; 0.001) were established among the control, LSIL and HSIL groups, while cyclin D1 showed significant statistical difference (p &lt; 0.05). Great variations were observed in nuclear morphology and nuclear surface area that had highly statistically significant differences (p &lt; 0.001) among the control, LSIL and HSIL groups. Conclusion. This study demonstrated that immunohistochemical analysis of p16, p63 and cyclin D1 are important for diagnosis of dysplastic changes in cervical epithelium. Also, morphometric analysis of the nuclear surface area demonstrated a high significance for diagnosis of cervical dysplasia.

Abstract 1236: In vitro association of chloroquine with alkylating and antimicrotubule agents in patient derived cell lines of glioblastoma

Abstract Autophagy is an important mechanism for cell survival after chemotherapy treatment. Many preclinical and clinical trials have tried to achieve tumor remission combining alkylating agents with autophagy inhibition. Chloroquine (CQ) has a long record as an autophagy inhibitor. In previous studies it was administered in vitro with mixed effects on central nervous system cell lines: while high doses of CQ were cytotoxic, low doses were neuroprotective. Preclinical studies showed that CQ could be used to treat glioblastoma, tumors derived from glial cells in the brain. In a phase I/II clinical trial combining CQ with the standard alkylating agent (temozolomide) plus radiotherapy treating patients with glioblastoma, a dose-limiting toxicity was observed at high doses. Although the authors reported that autophagy inhibition could not be achieved, they did not describe if this mechanism was involved in tumor resistance in that cohort of patients. In order to clarify if CQ can be used to block autophagy, we performed long term treatment combining low doses of CQ with chemotherapeutic agents. We used patient derived cell lines (PDCL) from glioblastoma tumors and treated them with therapeutic agents, using the reported plasma concentration of each drug. We measured acidic organelles with acridine orange incorporation 3, 5 and 7 days after addition of the treatment with flow cytometry. Additionally, 7 days after treatment surviving cells were counted. We did not observe significant increase in autophagy levels or decrease in cell number after temozolomide treatment. On the contrary, paclitaxel induced autophagy and decreased cell survival. Interestingly the combination of procarbazine, lomustine and vincristine (PCV), induced a broad range of results, ranging from high autophagy to increased cell death. We decided to combine PCV with CQ and perform long term analysis in order to evaluate the ability of cells undergoing or escaping apoptosis, senescence or mitotic catastrophe. After 5 days nuclear morphometric analysis demonstrated that out of 6 PDCL tested, only one presented an increase in senescence. The same PDCL also presented a decrease in total number of cells and increase in p62, although combining CQ with PCV was not better than PCV alone. Our results in vitro, using low concentrations of CQ corroborate the finding in clinical trials that chloroquine does not eliminate malignant cells in glioblastoma and better autophagy inhibitors need to be developed to achieve this goal. Citation Format: Franciele C. Kipper, Ajith Thomas, Guido Lenz. In vitro association of chloroquine with alkylating and antimicrotubule agents in patient derived cell lines of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1236.

Evaluation of the Effect of Matrine on Human Laryngeal Squamous Cell Carcinoma Cell Line

Aim of study: to evaluate the possible anticancer potential of Matrine, one of the main active components extracted from dry roots of Sophora flavescens Ait (Leguminosae), on human laryngeal squamous cell carcinoma cell line (Hep-2). Material and methods: Prepared (Hep-2) cell line was treated with different concentrations of Matrine for 48 hours. The effect of Matrine on cell line was investigated using MTT assay, cytological examination and nuclear morphometric analysis of treated cells to explore the effect of the drug on cell proliferation and induction of apoptosis. The results were analyzed statistically. P value ≤ 0.05 was considered significant. Results: MTT assay showed that the proliferation of Matrine treated cells (in relation to control cells) decreased in a dose dependent manner. Morphometric analysis showed a decrease in nuclear area factor (NAF), which is an indicator of apoptosis, as concentrations increased. The results were statistically significant. Conclusion: Matrine has a cytotoxic effect on Hep-2 cell line through induction of apoptosis.

Combined Cisplatin Treatment and Photobiomodulation at High Fluence Induces Cytochrome c Release and Cytomorphologic Alterations in HEp-2 Cells

BACKGROUND: Photochemotherapy is thought to be a novel therapeutic modality for cancer. The photobiomodulation (PBM), applied through high fluence low-level laser irradiation (HF-LLLI), can be combined with the chemotherapeutic drug cisplatin to gain the benefit of potentiating its cytotoxic effect at possibly lower doses.&#x0D; AIM: The study aimed at investigation of the apoptotic effect of PBM, through LLLI (at HF), alone and in combination with cisplatin on cultured laryngeal cancer (HEp-2) cells.&#x0D; MATERIALS AND METHODS: In the current experimental in vitro research, cultured laryngeal cancer cell line (HEp-2) was treated with the half maximal inhibitory concentration of cisplatin, with and without LLLI. The study design consisted of four groups: Control (untreated), cisplatin-alone-treated, PBM-alone-treated, and combination cisplatin + PBM treated groups. Cells were irradiated once with diode laser (wavelength 808 nm, energy output 350 mW, 3 min, fluence 190.91 J/cm2, and continuous wave mode). Cytotoxicity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and the potential apoptotic effect was evaluated by cytochrome c (CYC) release through enzyme-linked immunosorbent assay (ELISA), in conjunction with visualization of cytomorphologic alterations by light microscopic examination, followed by digital morphometric analysis of nuclear changes through estimation of nuclear area factor (NAF). Analysis of variance and post hoc multiple-comparison tests were used for statistical analysis of the data of cytotoxicity assay, ELISA, and nuclear morphometric analysis.&#x0D; RESULTS: PBM alone had a neutral effect on viability of HEp-2 cells, but it induced CYC release and lowered NAF mean value, significantly. When PBM was combined with cisplatin, more conspicuous deterioration in bioavailability of HEp-2 cells was observed, a higher amount of CYC was liberated and NAF value dropped in HEp-2 cells, compared to those which received separate treatments with cisplatin alone or PBM alone.&#x0D; CONCLUSION: Based on the current findings, low-level laser photochemotherapy might be a promising adjunctive anticancer treatment for laryngeal cancer, as PBM at HF was able to augment the apoptotic effect of cisplatin on HEp-2 cancer cells.

Nuclear morphometry in indeterminate thyroid nodules.

Up to 30% of thyroid nodules undergoing fine needle aspiration (FNA) yield an indeterminate result. Recent research efforts have suggested that nuclear morphometry and morphology may enhance the diagnostic accuracy of FNA as an objective adjunct. We applied nuclear morphometric analysis on a diverse cohort of patients to evaluate the association between nuclear morphometry and malignancy. Forty-five randomly selected patients, who underwent thyroid surgery after an indeterminate FNA result (Bethesda III & IV) between 2012-2015, were reviewed. One hundred representative nuclei per FNA of a thyroid nodule were analyzed using ImageJ. Seven validated morphometric parameters were collected: nuclear area, perimeter, circularity, aspect ratio, roundness, and maximum/minimum Feret's diameter. L/S ratio was subsequently calculated. All 8 nuclear parameters were reported as averages with standard errors of the mean (SEM). A Student's t-test was used to assess the association of nuclear parameters with final surgical pathology. The mean age of all patients was 56.31±15.39 years, with female patients comprising 68.9% of the cohort. Twenty-two patients had malignant thyroid nodules. The mean perimeter of nuclei for the cohort was 18.48±0.45 µm, the mean area was 22.19±0.93 µm, and the mean maximum Feret's diameter was 6.67±0.13 µm. No significant differences in the 8 nuclear parameters were observed between the malignant and non-malignant groups. In the population examined, our results suggest that nuclear morphometry is not yet a tool of reliable diagnostic value in accessing malignant and non-malignant thyroid nodules. Further investigation is necessary to identify objective parameters that will enhance diagnostic accuracy of indeterminate FNA cytology to minimize the number of diagnostic thyroid surgery.

Nuclear morphometric analysis in tissue as an objective tool with potential use to improve melanoma staging.

Alterations in nuclear size and shape are commonly observed in cancers, and its objective evaluation may provide valuable clinical information about the outcome of the disease. Here, we applied the nuclear morphometric analysis in tissues in hematoxylin and eosin-digitized slides of nevi and melanoma, to objectively contribute to the prognostic evaluation of these tumors. To this, we analyzed the nuclear morphometry of 34 melanomas classified according to the TNM stage. Eight cases of melanocytic nevi were used as non-neoplastic tissues to set the non-neoplastic parameters of nuclear morphology. Our samples were set as G1 (control, nevi), G2 (T1T2N0M0), G3 (T3T4N0M0), G4 (T1T2N1M1), and G5 (T3T4N1M1). Image-Pro Plus 6.0 software was used to acquire measurements related to nuclear size (variable: Area) and shape (variables: Aspect, AreaBox, Roundness, and RadiusRatio, which were used to generate the Nuclear Irregularity Index). From these primary variables, a set of secondary variables were generated. All the seven primary and secondary variables related to the nuclear area were different among groups (Pillai's trace P<0.001), whereas Nuclear Irregularity Index, which is the variable related to nuclear shape, did not differ among groups. The secondary variable 'Average Area of Large Nuclei' was able to differ all pairwise comparisons, including thin nonmetastatic from thin metastatic tumors. In conclusion, the objective quantification of nuclear area in hematoxylin and eosin slides may provide objective information about the risk stratification of these tumors and has the potential to be used as an additional method in clinical decision making.

Nuclear Morphometric Analysis in Fine Needle Aspirates of Breast Neoplasms

Nuclear Morphometric Analysis in Fine Needle Aspirates of Breast Neoplasms

Vinblastine and antihelmintic mebendazole potentiate temozolomide in resistant gliomas.

Glioblastoma (GBM) is a very aggressive tumor that has not had substantial therapeutic improvement since the introduction of temozolomide (TMZ) in combination with radiotherapy. Combining TMZ with other chemotherapeutic agents is a strategy that could be further explored for GBM. To search for molecular predictors of TMZ resistance, the TCGA (The Cancer Genome Atlas) database was utilized to assess the impact of specific genes on TMZ response. Patients whose tumors expressed low levels of FGFR3 and AKT2 responded poorly to TMZ. Combination treatment of vinblastine (VBL) plus mebendazole (MBZ) with TMZ was more effective in reducing cell number in most cultures when compared to TMZ alone, especially in cells with low expression levels of FGFR3 and AKT2. Cell cycle distribution and nuclear morphometric analysis indicated that the triple combination of TMZ, VBL and MBZ (TVM) was able to induce polyploidy and senescence, in addition to increasing the Notch3 RNA level in patient-derived gliomas. Thus, this set of data suggests that the triple combination of TMZ, VBL and MBZ may be a considerable therapeutic alternative for the TMZ-tolerant gliomas that harbor low expression of FGFR3/AKT2.

Doxazosin nanoencapsulation improves its in vitro antiproliferative and anticlonogenic effects on breast cancer cells

Doxazosin has been evaluated for the treatment of several types of cancer. Here, the antitumor effect of the nanoencapsulated form of doxazosin was evaluated in an in vitro model of breast cancer (MCF7 cell line). Doxazosin-loaded polymeric nanocapsules (DXZ-NC) were produced by interfacial deposition of preformed polymer with homogeneous aspect, spherical shape, mean diameter of about 130nm, positive zeta potential (+5mV), and encapsulation efficiency close to 35%. The Alamar Blue® assay and cell counting were carried out to assess cell viability and cell number, respectively. Mechanism of death was evaluated by Annexin/Propidium Iodide staining, while the long-term response was assessed using the clonogenic assay. Nuclear morphometric analysis was investigated using the NMA technique. A significant decrease in cell viability and clonogenicity was observed after the treatment with DXZ-NC when compared to the non-encapsulated drug. All treatments induced apoptosis as the main mechanism of toxicity. In conclusion, the nanoencapsulation of doxazosin improved its in vitro effects in MCF7 cells, without changing the mechanism of cell death underlying its toxicity. This approach was fundamental to reduce the long-term in vitro ability of the remaining tumor cells to form new colonies after the treatment, potentially reducing the risk of tumor recurrence.