Proliferating Cell Nuclear Antigen Index Research Articles

Pathomorphological and Immunohistochemical Study of Fibrosarcoma in an Adult Dog: A Case Report

Aim: The objective of the present case report is to describe pathomorphological findings and immunohistochemical expression of pancytokeratin (PCK), vimentin, proliferating cell nuclear antigen (PCNA) and Ki67 in a case of fibrosarcoma in dog. Case Presentation: A large, round ball-like growth of approximately 14 centimetres in diameter was noticed on caudal aspect of left thigh region in ten years old male bull dog. Its surface was ulcerated. It was soft in consistency and cut surface appeared pinkish-white. Giemsa-stained smears revealed spindle cells with elongated nuclei containing granular chromatin, single to multiple nucleoli and scanty cytoplasm. Histopathological examination revealed moderately to well-differentiated neoplastic fibroblasts arranged in interwoven pattern with the presence of collagenous stroma. Neoplastic cells were characterized by pleomorphic, oval to elongated nuclei with single to multiple prominent nucleoli and moderate to scanty cytoplasm. Mitotic count was 1.2/high power field. The Immunohistochemical staining revealed intracytoplasmic positive immunoreactivity for vimentin and no immunoreactivity for PCK. Proliferating neoplastic cells revealed mild to strong nuclear immunostaining for PCNA and Ki67 with 56.7% PCNA index and 4.0% Ki67 index. Conclusion: Based on histopathological findings, the case was diagnosed as fibrosarcoma. Immunopositive reactivity for vimentin and no immunoreactivity for PCK indicated the mesenchymal origin of the tumour. Histopathological features, low mitotic count and low Ki67 index suggested the less aggressive behaviour of the tumour in the present case.

Prognostic value of proliferation markers Ki-67 and PCNA in esophageal squamous cell carcinoma.

Ki-67 and proliferating cell nuclear antigen (PCNA) are markers of proliferation used to assess the growth fraction of the cell population. The present study aimed to explore the prognostic value of these proliferative markers in patients with resected esophageal squamous cell cancer (ESCC) in a large cohort. A total of 807 patients with ESCC who underwent radical resection were retrospectively reviewed. Ki-67 and PCNA index were examined as the percentage of positively nuclear-stained cells among total number of cancer cells in three high-power fields by a pathologist who was blinded to the patients' history and outcome. Overall survival (OS) and disease-free survival (DFS) were estimated. The Cox regression model was used to evaluate the independent factor. The cut-off value as 60 and 80% for Ki-67 and PCNA were verified, respectively. Higher Ki-67 expression was associated with low differentiation and more lymph node metastasis. Higher PCNA expression was associated with elevated T stage. However, either expression of Ki-67 or PCNA was not correlated with OS and DFS. While in combination of Ki-67 and PCNA analysis, higher expression of these two proliferative markers predicted worse prognosis (median OS, 47 months versus 54 months, P = 0.04). Whatever the combined proliferative marker, differentiation, lymph node metastasis stage and vascular invasion act as factors in univariate survival analysis, but combined Ki-67 and PCNA is not an independent prognostic variable in multivariate analysis (P = 0.10). Our results suggest that proliferative markers of Ki-67 and PCNA may correlate with tumor stage but cannot act as independent predictor of prognosis in ESCC patients.

The number of the intraepithelial T cells correlate with the proliferation index in human bulbourethral gland epithelium

BackgroundOur study has immunohistochemically examined T cells localization and number as well as proliferative activity level for the bulbourethral gland epithelium in men of different ages, using monoclonal antibodies against CD45RO and proliferating cell nuclear antigen (PCNA). ResultsWe have found that the T cells have been localized mainly in excretory ducts epithelium of the glands in any age group, meanwhile their relative number varies with age. The excretory ducts epithelium has shown a high proliferative activity when in acini the PCNA index has been low. Postnatal dynamics of the epithelium proliferative activity positively correlates with age-related density fluctuations in lymphocytic infiltration of the glands. ConclusionsWe consider that intraepithelial T cells may contribute to the regulation of epithelial cells proliferation in the bulbourethral glands.

Markers of tumor cell proliferation in cancerous tumors of the larynx

The cell proliferative process in the larynx cancer is studied. Contrary to the classical mitotic index the Ki-67 and PCNA indices are reliable and reproducible on the clinical material by the proliferative cell activity indices of the larynx tumors. There is a reverse connection between the proliferation index in cells of the squamous larynx cancer and the tumor differentiation. The direct correlation of the cell proliferation index of the squamous laxynx cancer with the disease stage is revealed. The preoperative radiation therapy significantly decreases the cell proliferation rates of the squamous larynx cancer.

Immunohistochemical analysis of proliferating cell nuclear antigen and minichromosome maintenance complex component 7 in benign and malignant salivary gland tumors.

Background:Proliferation markers have been used to determine the behavior and prognosis of benign and malignant tumors; this study was aimed to compare the immunohistochemical (IHC) expression of proliferating cell nuclear antigen (PCNA) and novel marker minichromosome maintenance complex component 7 (MCM7) in common salivary gland tumors including pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (AdCC), to find a possible significant correlation between benign and malignant tumors.Materials and Methods:In this cross-sectional study, a total of 90 cases, including 30 PAs, 30 MECs, and 30 AdCCs, were collected. The IHC expressions of PCNA and MCM7 were evaluated. Their expressions were compared with each other and between benign and malignant tumors. Statistical analysis was performed by Chi-square and Tukey's test. P value was considered 0.05.Results:Out of 30 cases of PA, 28 cases (93.3%) were PCNA positive and 28 cases (93.3%) were MCM7 positive. In the AdCC cases, 29 cases (96.6%) were PCNA positive and 29 cases (96.6%) were MCM7 positive. In the MEC cases, all cases (100%) were PCNA positive and 23 cases (76.6%) were MCM7 positive. The labeling index (LI) of MCM7 and PCNA was evaluated, and this index was lower in MCM7 LI than PCNA in all tumors. The MCM7 and PCNA expression showed a significant difference in PA and MEC (P < 0.001).Conclusion:PCNA expression was higher than MCM7 expression in salivary gland tumors. However, more studies are needed to evaluate the malignant activity of these tumors with group of markers such as MCM family members.

Adiponectin Prevents Restenosis Through Inhibiting Cell Proliferation in a Rat Vein Graft Model.

ResumoFundamento:O enxerto de bypass na artéria coronária (CABG) continua a ser eficiente como tratamento para pacientes portadores de doença arterial coronariana; entretanto, o enxerto venoso tende a apresentar reestenose ou oclusão. A adiponectina (ADP) é uma proteína hormonal plasmática com a função de regular a proliferação celular.Objetivo:Foram utilizadas duas doses diferentes da proteína ADP em um modelo de enxerto venoso em ratos para estimular a alteração do enxerto venoso. O objetivo deste estudo foi investigar o efeito da ADP sobre a reestenose em enxerto venoso.Métodos:Veias jugulares autólogas foram implantadas como enxertos interposicionais de carótida pela técnica de anastomose de manga em ratos Sprague Dawley. A adiponectina (2,5 μg e 7,5 μg) foi entregue ao enxerto venoso por bypass de forma perivascular, suspensa em gel Pluronic-F127 a 30%. O grupo tratado apenas com bypass e o grupo tratado com gel veículo carregado apenas com Pluronic funcionaram como controle. Foram feitas comparações com análise de via única de variância e teste post-hoc, com p <0,05 sendo considerado significativo.Resultados:A proliferação celular (índice de PCNA) foi significativamente baixa no grupo tratado com adiponectina em comparação com o grupo de controle e o grupo tratado com o gel veículo na íntima e na adventícia dos enxertos a partir do dia 3 (p <0,01). VCAM-1 e ICAM-1 avaliados por imuno-histoquímica diminuíram significativamente em enxertos venosos tratados com adiponectina na quarta semana (p <0,01). O tratamento de enxertos venosos com gel carregado com adiponectina reduziu a espessura da íntima, da média e da adventícia, em comparação com os enxertos de controle e tratados com gel veículo no dia 28 (p <0,01).Conclusões:Este estudo oferece evidências adicionais do possível papel terapêutico da adiponectina na modulação de lesão vascular e seu reparo.

Oral glutamine inhibits tumor growth of gastric cancer bearing mice by improving immune function and activating apoptosis pathway

Gastric cancer is one of the most common cancers in the world. It has been shown that exogenous glutamine (GLN) can inhibit the growth of tumor in vivo, but the relationship between GLN and gastric cancer has not been studied. The gastric cancer bearing mouse model was constructed and taken GLN orally at the same time, and the results found that oral GLN (1 or 2 g/kg/d) significantly inhibited the growth rate of tumor and reduce the weight of tumor tissues. Immunohistochemistry showed that oral GLN significantly reduced the PCNA index, which further proved that GLN could inhibit the growth of tumor cells. At the same time, TUNEL assay showed that oral GLN significantly enhanced the apoptosis levels of tumor cells. In addition, GLN reduced GSH levels in tumor tissues, but increased the levels of GSH in plasma, improved the T-lymphocyte transformation rate and NK cell activity, significantly inhibited the secretion of TNF-α and promoted the secretion of IL-2, thus regulating the immune function in vivo. Further detection of apoptosis pathway showed that oral GLN significantly enhanced the expression of pro-apoptotic factor Bad and inhibited the expression of Bcl-2. Meanwhile, GLN significantly increased the activities of Caspase-3, Caspase-8, caspase-9 and PARP. GSH activator NAC had a similar effect to GLN, which could improve the immune function and activate apoptosis pathway, while GSH inhibitor BSO significantly blocked the regulation of GLN, destroyed the immune balance and inhibited apoptosis, but IL-2 significantly blocked the anti-apoptotic effect of BSO. Therefore, oral GLN can improve immune function and activate apoptosis pathway through GSH, and then inhibit the growth of tumor in vivo.

Abstract 14: TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model

Abstract Colorectal cancer (CRC) is a major health problem globally. Preventing CRC in the general population, and especially in high-risk cohorts with colonic polyps, or its recurrence, is a high priority. ONC201, an orally active small molecule with robust antitumor activity, is currently in clinical trials for advanced cancers. The aim of this study was to determine the CRC preventive potential of ONC201 using the Apcmin/+ mouse model for CRC in familial adenomatous polyposis. First, the optimal and non-toxic doses of ONC201 were determined. C57BL/6J mice (n=6/group) were randomized, and five different doses of ONC201 (0-100mg/kg BW) were administered orally twice a week by gavage. After six weeks of treatment, all mice were euthanized, and tissues were analyzed for optimal dose and toxicity. Based on the body weight gain, gross organ weights at termination, blood profiling, plasma liver enzyme profile, and histopathological analysis, it was concluded that there were no toxicities associated with ONC201 within the tested dose range. For efficacy evaluation, Apcmin/+ male and female mice (n≥20) were grouped and ONC201 (0, 25 and 50 mg/kg BW) was administered by gavage, twice weekly for 14 weeks. At termination, intestinal tumors were evaluated for incidence and multiplicity to determine the efficacy of ONC201. Results indicate a strong suppressive effect of ONC201 against colonic and small intestinal (SI) tumor multiplicity in mice of both genders. Colonic tumor incidence was significantly reduced by &amp;gt;50% in ONC201-treated (50 mg/kg BW) male (40% in treated vs. 92% in control; p&amp;lt;0.0002) and female mice (30% in treated vs. 81% in control; p&amp;lt;0.001) when compared with placebo. Colonic tumor multiplicity was reduced by 68% in ONC201-treated (50 mg/kg BW) male mice (0.44 ± 0.11 in treated vs. 1.4 ± 0.14 in control; p&amp;lt;0.0001) and by 75% in female mice (0.30 ± 0.10 in treated vs. 1.19 ± 0.19 in control; p&amp;lt;0.0003). SI polyps were suppressed by up to 68% in both male mice (11.40 ± 1.19 in treated vs. 36.08 ± 2.62 in control; p&amp;lt;0.0001) and female mice (9.65 ± 1.15 in treated vs. 29.24 ± 2.51 in control; p&amp;lt;0.0001). Snap-frozen colon tumor samples were analyzed for various biomarkers. Western blot protein analysis of the tumors suggested a dose-dependent increase in TRAIL expression upon treatment with ONC201. Similar increases were observed for DR5 and cleaved caspase 8 protein expression. Cleaved caspase 7, caspase 3, FADD and p21 were found to be increased in the treated tumor samples when compared with control. Immunohistochemical analysis indicated a significant decrease in PCNA index in the ONC201-treated tumors compared with controls. Serum analysis indicated a decrease in pro-inflammatory biomarkers, such as IL1β, IL6, TNFα, G-CSF, and GM-CSF, in the ONC201-treated samples compared to controls. Overall, chronic oral administration of ONC201 demonstrated strong chemopreventive efficacy against intestinal tumorigenesis in the Apcmin/+ mouse model. (Supported by NCI HHSN 261201500038I) Citation Format: Chinthalapally V. Rao, Venkateshwar Madka, Yuting Zhang, Gopal Pathuri, Janani Panneerselvam, Nicole Stratton, Niklas K. Finnberg, Howard P. Safran, Jennifer Fox, Shizuko Sei, Elizabeth R. Glaze, Robert Shoemaker, Wafik S. El-Deiry. TRAIL inducing small molecule ONC201 prevents intestinal tumors in FAP mouse model [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 14.

Abstract 13: Aspirin and metformin combination inhibits PDAC progression and metastasis in KPC mice

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that generally escapes detection until it reaches an advanced stage. Hence, developing strategies that inhibit PDAC progression is of utmost importance. Observational studies suggest that frequent use of metformin and aspirin reduces PDAC risk. Since these agents have different mechanisms of action, combining them may lead to additive inhibitory effects on PDAC. Here we aimed to validate the usefulness of the combination of aspirin with metformin in the Ptf1acre-ERTM.LSL-KrasG12D/+. LSL-Trp53R172H/+ (KPC) mouse model. Aspirin (600 ppm or 1,200 ppm) and metformin (500 ppm or 1,000 ppm) either individually or in combination were tested for their effect on pancreatic intraepithelial neoplasia (PanINs), PDAC, and metastasis in KPC mice. Eight week-old KPC mice were administered tamoxifen by gavage to initiate tumorigenesis (PanINs) and randomized to control and experimental groups. Seven weeks later, experimental diets containing test agents individually or in combination were administered to the KPC mice. Mice were euthanized 32 weeks post-tamoxifen administration and pancreata were evaluated. Histological analysis showed 100% penetrance of PanIN lesions in all the KPC mice. Control diet fed mice showed 100% PDAC incidence. Aspirin (1,200 ppm) or metformin (1,000 ppm) showed significant inhibition of PDAC incidence by 15.6% (P&amp;lt;0.03) and 23.5%(P&amp;lt;0.0007), respectively, while the combination of aspirin (600 ppm or 1,200 ppm) with metformin (1,000 ppm) also showed significant 20% (P&amp;lt;0.02) to 23% (P&amp;lt;0.02) inhibition of PDAC incidence. Metastasis incidence was observed in 69% KPC mice fed the control diet. Importantly, KPC mice fed 1,000 ppm metformin showed only 21% metastasis incidence (P&amp;lt;0.002). Also, combination of aspirin (600 ppm or 1,200 ppm) with metformin (500 ppm or 1000 ppm) inhibited metastasis by 40% - 56% (P&amp;lt;0.07-P&amp;lt;0.007). Tumor necrosis was observed in 61% of control mice that was also significantly reduced by 73% - 100% (P&amp;lt;0.004 - P&amp;lt;0.0001) in treated mice. There was also an inhibition of PanINs by aspirin (43.8%; p&amp;lt;0.02) or metformin (35.7%; P&amp;lt;0.034) alone when compared with control mice. Biomarker analysis suggested that treatment with aspirin and/or metformin showed a significant inhibition of PCNA index (P&amp;lt;0.05- P&amp;lt;0.0001). In spite of aggressive tumor progression in KPC mice, aspirin and/or metformin showed inhibitory effects on PDAC incidence, metastasis, and necrosis; however, combinations failed to show any additive or synergistic effect when compared with 1,000 ppm metformin alone treatment. (Supported by NCI PREVENT Program HHSN261201200013I/ HHSN26100010) Citation Format: Altaf Mohammed, Venkateshwar Madka, Gaurav Kumar, Anil Singh, Nicole Stratton, Stanley Lightfoot, Mark S. Miller, Chinthalapally V. Rao. Aspirin and metformin combination inhibits PDAC progression and metastasis in KPC mice [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 13.

The effect of elemene in combination with vascular endothelial growth factor polyclonal antibody on the expression of proliferating cell nuclear antigen and tissue inhibitor of metalloproteinase-1 in brain glioma tissue of rats

Objective To investigate the effect of elemene in combination with vascular endothelial growth factor (VEGF) polyclonal antibody on the expression of proliferating cell nuclear antigen (PCNA) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in brain glioma tissue of rats. Methods The rat glioma models were built by intracerebral injection of C6 glioma cells. Forty-eight successful rat models were selected and randomly divided into model group, elemene group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group, with 12 rats in each group. Rats in model group were performed intraperitoneal injection of 0.5 ml normal saline, rats in elemene group were performed intraperitoneal injection of 0.5 ml elemene injection, rats in VEGF polyclonal antibody group were performed intraperitoneal injection of 0.5 ml VEGF polyclonal antibody, and rats in elemene in combination with VEGF polyclonal antibody group were performed intraperitoneal injection of 0.5 ml elemene injection and 0.5 ml VEGF polyclonal antibody. The drug was administered on alternate days for 7 consecutive times. The tumour volume, tumor inhibition rate and the expression of PCNA and TIMP-1 in brain tissue of the rats between the groups were compared. Statistics with SPSS 21.0 software, measurement data for Mean±SD indicated that one factor variance analysis was used for the comparison between the two groups, and t test was used for the comparison between the two groups. Results (1) Tumor volume and tumor inhibition rate: The tumor volumes in model group, elemene group, polyclonal group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group were (68.54±8.12), (37.65±6.24), (39.12±6.41), (30.12±5.24) mm3, respectively; the tumor inhibition rates in elemene group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group were (445.07±6.45)%, (42.92±6.52)%, (56.05±7.21)%, respectively. The tumor volume in elemene in combination with VEGF polyclonal antibody group was significantly smaller than that in control group (t=10.449, 9.851, 13.772, P<0.01); the tumor volume in elemene in combination with VEGF polyclonal antibody group was significantly smaller than that in elemene group and VEGF polyclonal antibody group (t=3.201, 3.766, P<0.05), and the tumor inhibition rate was significantly higher than that in elemene group and VEGF polyclonal antibody group (t=3.932, 4.679, P<0.05). (2) As to the expression of PCNA and TIMP-1 in brain tissue: the PCNA indexes of tumors in model group, elemene group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group were (73.12±10.21, 44.24±6.42, 47.36±7.12, 37.45±5.24), respectively, and the TIMP-1 indexes were (45.24±6.45, 62.45±8.15, 59.45±7.32, 78.65±11.24), respectively. The PCNA indexes of tumors in elemene group, VEGF polyclonal antibody group and elemene in combination with VEGF polyclonal antibody group were significantly lower than that in model group (t=8.295, 7.447, 10.767, P<0.05, P<0.01), the TIMP-1 indexes were significantly higher than that in model group (t=5.736, 5.401, 8.931, P<0.05, P<0.01); the PCNA index of tumor in elemene in combination with VEGF polyclonal antibody group was significantly lower than that in elemene group and VEGF polyclonal antibody group (t=2.838, 3.491, P<0.05), and the TIMP-1 index was significantly higher than that in elemene group and VEGF polyclonal antibody group (t=4.042, 4.700, P<0.05). Conclusion Elemene in combination with VEGF polyclonal antibody can help to inhibit the growth of brain glioma tissue, which may be related to the regulation of PCNA and the expression of TIMP-1 in brain tissue. Key words: Brain glioma; Elemene; Vascular endothelial growth factor polyclonal antibody; Proliferating cell nuclear antigen; Tissue inhibitor of metalloproteinase-1

Decellularized matrix of human fatty liver used for three-dimensional culture of hepatocellular carcinoma cells

To construct a decellularized matrix of human fatty liver as the scaffold for three-dimensional (3D) culture of hepatocarcinoma cells. Human fatty liver decellularized matrix (hFLM) was prepared by repeated freezingthawing, perfusion with gradient SDS and 1% Triton X-100 through the portal vein and hepatic artery, and repeated agitation with Triton X-100. HepG2 cells were cultured in the prepared hFLM, and the cell survival, morphology, proliferation and cellular expressions of the adhesion molecules were detected. The decellularization procedure shortened the time for scaffold preparation and preserved the 3D ultrastructure and the composition of the extracellular matrix. HepG2 cells cultured in hFLM scaffold maintained proliferation for up to 15 days and showed a growth pattern with a long lag phase and a slow growth rate, which was similar to the growth pattern in vivo. The cultured HepG2 exhibited a low expression of E-cadherin and a high expression of vimentin, which was consistent with the xenograft but opposite to 2D cultured cells. However, the lack of adequate nutrient transport in this hepatocarcinoma cell model led to a slowdown of cell proliferation in the later stage. The PCNA index of HepG2 cells cultured in hFLM was lowered by 29.3% on day 12 as compared with that on day 6. We established a new protocol for preparing hFLM and confirmed the feasibility of constructing hepatocarcinoma cell models using the hFLM scaffold.

Adipose-derived stem cells-induced burn wound healing and regeneration of skin appendages in a novel skin island rat model.

The study of effectiveness of adipose-derived stem cells (ASCs) in treating burn wounds is still a developing field. The process of wound contraction in areas of loose skin is a major confounding factor in the evaluation and study of burn wound healing in animal models. To evaluate the effect of local ASCs administration, deep partial thickness burn wounds were induced by 30 s application of hot copper plates in a novel skin island burn wound rat model to avoid interference from primary wound contraction. Skin islands were divided into two treatment groups-control group (n = 9) injected with PBS and ASCs-treated group (n = 9) injected with 5 × 10 ASCs intradermally. Progress in wound healing was checked at regular intervals after injury (on 1st, 2nd, 3rd, and 4th week) by measuring the mean wound area and analyzing the wound histologically and immunohistochemically, after unstitching the overlaying skin to expose the skin island. It was found that local intradermal injection of ASCs improved burn wound healing at all given time points when compared with control groups, especially in the first 2 weeks (p < 0.05). The percentage of live follicles increased gradually in the ASCs-treated groups compared with control groups between the 3rd and 4th weeks (p < 0.05). The vascular density and proliferating cell nuclear antigen index were also significantly increased in the ASCs-treated groups. Thus, in this study, a novel burn wound rat model with reduced interference from wound contraction has been put forth to investigate the therapeutic effects of local administration of ASCs on burn wound healing. Local injection of ASCs not only improved burn wound recovery but also enhanced angiogenesis and skin appendage regeneration.

Antioxidant and anti-apoptotic effects of royal jelly against nicotine-induced testicular injury in mice.

This study describes the effects of royal jelly (RJ) on testicular injury induced by nicotine (NIC) in mice. Thirty-six male BALB/c mice were randomly divided into six groups (n = 6). Group 1 received normal saline, group 2 received 100 mg/kgBW/day RJ, groups 3 and 4 received NIC at doses of 0.50 and 1.00 mg/kgBW/day, respectively, and groups 5 and 6 received NIC at doses of 0.50 and 1.00 mg/kg BW/day, respectively, plus RJ. Following 35 days, the serum level of testosterone, histopathological changes, germ cell apoptosis, proliferating cell nuclear antigen (PCNA), malondialdehyde (MDA) content, and antioxidant indexes including total antioxidant capacity (TAC) and catalase (CAT) activity were determined. In addition, the mitochondria-dependent apoptosis was investigated by assessing the Bcl-2, p53, and Caspase-3 mRNA levels expression by reverse transcription-PCR (RT-PCR). Compared to NIC receiving groups, the concomitant administration of RJ could protect the testosterone reduction and histological damages. After RJ treatment, the level of tissue MDA content decreased, while tissue TAC and CAT levels were remarkably increased compared to NIC-exposed groups. Remarkable higher TUNEL-positive germ cells and low PCNA index were observed in NIC receiving groups. Besides, the expression level of Bcl-2 was significantly higher and the p53 and Caspase-3 levels were significantly lower in the RJ co-administration groups than NIC-only receiving groups. Our results confirmed that RJ effectively protects the testis against NIC evoked damages by antioxidant and anti-apoptotic effects involving the up regulation of the antioxidant status, mitochondria-dependent apoptosis pathway prevention, and the proliferating activity improvement.

Multistep carcinogenesis of 7,12-Dimethylbenz(a)anthracene (DMBA)-induced skin tumours in rats: An immunohistochemical study

The present study was aimed to induce skin tumours in rats by chemical carcinogen 7,12-dimethybenz(a)anthracene (DMBA), to ascertain the different stages of tumorigenesis with molecular markers and application of these markers in diagnosis of neoplasms. DMBA solution (5 pmol in 0.25 ml acetone per dose) was applied fortnightly on the skin of 35 rats in one group (Group A) for a maximum of 20 doses. In another group of 35 rats (Group B), DMBA was applied initially for three consecutive weeks as initiator followed by weekly croton oil (1% w/v in acetone) applications as promoter for 60 weeks. Control groups (10 rats each) were also taken for both the treatment groups. Skin tumours were induced in only DMBA treated rats after latent period of 22 weeks, comprising of papillomas (47%), squamous cell carcinomas (43%) and fibrosarcomas (13%). Adnexal tumours consisted only two cases, one each of sebaceous basal cell carcinoma and sweat gland adenocarcinoma. Tumours arose as soft sessile/pedunculated, multicentric growths which became large cauliflower like and multiple confluencing lesions to become bigger masses. Histopatho- logically, sequential alterations seen were epidermal hyperplasia, severe epidermal hyperplasia, papilloma, papillary carcinoma, squamous cell carcinoma and its metastasis in lungs. The indices for proliferation markers (mitoses, AgNORs and PCNA) were significantly higher for squamous cell carcinoma (9.14, 15.44 and 181.63) and papillary carcinoma (3.09, 6.40 and 79.13) compared to mild epidermal hyperplasia (1.03, 4.17 and 0.53). Immunohistochemically, epidermal cells revealed p53, c-Myc and PCNA positive nuclear labelling for initiation and proliferation; strongly positive labelling for pre-malignant lesions, and strong p53, c-Myc, PCNA and hTERT labelling for malignant transformation, and additional abnormal cadherin and fibronectin for invasion and metastasis. c-Myc oncoprotein was demonstrated in nucleus and cytoplasm of cancer cells by immuno-electron microscopy. In conclusion, different stages of tumour development and progression was ascertained by using molecular markers: early detection of p53 and c-Myc for cell initiation and transformation; PCNA for cell proliferative activity; p53, c-Myc and PCNA overexpression in many cells for pre-malignant lesions; excessively increased p53, c-Myc and PCNA with additional expression of hTERT and abnormal cadherin and fibronectin for invasiveness, and additional loss of cadherin and fibronectin for metastasis. Mitotic index, AgNOR counts and PCNA index were independent cell proliferation markers, each being equally capable of ranking neoplastic lesions as benign and/or malignant tumours. Overall, PCNA index had high sensitivity and reliability for differentiating borderline tumours (papillary carcinomas) from benign (papilloma). Further studies are needed to elucidate the functional differences of excessively expressed, stabilized normal p53 protein and/or mutant p53 protein vis-a-vis their ultimate impact in the oncogenesis. Role of cytoplasmic accumulation of c-Myc in malignant tumours, particularly in malignant differentiated cells with the possibility of exploitation of this property of c-Myc oncoprotein for diagnosis of cancers needs further investigation.

Comparative pulmonary toxicity of a DWCNT and MWCNT-7 in rats

Very little is known about the in vivo toxicity of inhaled double-walled carbon nanotubes (DWCNTs). In the present study, we compared the pulmonary toxicity of DWCNT to MWCNT-7, a well-known multi-walled carbon nanotube. Rats were divided into six groups: untreated, vehicle, low-dose DWCNT, high-dose DWCNT, low-dose MWCNT-7, and high-dose MWCNT-7. The test materials were administered by intra-tracheal intra-pulmonary spraying (TIPS) every other day for 15 days: the low-dose and high-dose groups were administered final total doses of 0.25 and 0.50 mg/rat of the test material. The animals were sacrificed 1 and 6 weeks after the final TIPS administration. Six weeks after the final TIPS administration, rats administered MWCNT-7 had high levels of macrophage infiltration into the lung with dense alveolar wall fibrous thickening throughout the lung; significant elevation of lactate dehydrogenase activity, alkaline phosphatase activity, and total protein concentration in the bronchioalveolar lavage fluid; an increase in the pulmonary cell PCNA index; slightly elevated levels of 8-OHdG DNA adducts in lung tissue DNA; a small but significant increase in protein concentration in the pleural cavity lavage fluid and an increase in the visceral mesothelial cell PCNA index. None of these parameters was increased in rats administered DWCNT. The primary lesion in rats administered DWCNT was scattered formation of granulation tissue containing internalized DWCNT fibers. Our data indicate that DWCNT has lower pulmonary and pleural toxicity than MWCNT-7.

Potassium octatitanate fibers induce persistent lung and pleural injury and are possibly carcinogenic in male Fischer 344 rats.

Potassium octatitanate fibers (K2O·8TiO2, POT fibers) are widely used as an alternative to asbestos. We investigated the pulmonary and pleural toxicity of POT fibers with reference to 2 non‐fibrous titanium dioxide nanoparticles (nTiO 2), photoreactive anatase (a‐nTiO 2) and inert rutile (r‐nTiO 2). Ten‐week‐old male F344 rats were given 0.5 mL of 250 μg/mL suspensions of POT fibers, a‐nTiO 2, or r‐nTiO 2, 8 times (1 mg/rat) over a 15‐day period by trans‐tracheal intrapulmonary spraying (TIPS). Rats were killed at 6 hours and at 4 weeks after the last TIPS dose. Alveolar macrophages were significantly increased in all treatment groups at 6 hours and at 4 weeks. At week 4, a‐nTiO 2 and r‐nTiO 2 were largely cleared from the lung whereas a major fraction of POT fibers were not cleared. In the bronchoalveolar lavage, alkaline phosphatase activity was elevated in all treatment groups, and lactate dehydrogenase (LDH) activity was elevated in the a‐nTiO 2 and POT groups. In lung tissue, oxidative stress index and proliferating cell nuclear antigen (PCNA) index were elevated in the a‐nTiO 2 and POT groups, and there was a significant elevation in C‐C motif chemokine ligand 2 (CCL2) mRNA and protein in the POT group. In pleural cavity lavage, total protein was elevated in all 3 treatment groups, and LDH activity was elevated in the a‐nTiO 2 and POT groups. Importantly, the PCNA index of the visceral mesothelium was increased in the POT group. Overall, POT fibers had greater biopersistence, induced higher expression of CCL2, and provoked a stronger tissue response than a‐nTiO 2 or r‐nTiO 2.

Influence of long-term, high-dose dexamethasone administration on proliferation and apoptosis in porcine hepatocytes

The aim of this study was to examine the influence of long-term, high-dose dexamethasone administration on the liver, with particular emphasis on hepatocyte proliferation and apoptosis, using a swine model. The study included 48 large, female Polish breed pigs aged 3months (weighing ca. 30kg) divided into groups I (control; n=24) and II (dexamethasone; n=24) that receiving intra-muscular injections of monosodium phosphate dexamethasone for 29days. The pigs were euthanized on days subsequent to the experiment. Immediately after the euthanasia, the pig livers were sampled, fixed, and processed routinely for histopathology, histochemistry, and immunohistochemistry (for proliferating cell nuclear antigen, Bcl-2, and caspase-3). Apoptosis was visualized by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL). Dexamethasone administration gradually caused hepatocyte glycogen degeneration and finally lipid degeneration, accompanied by sinusoid and central vein dilatation and nuclear chromatin condensation. The proliferating cell nuclear antigen index, mean number of argyrophilic nucleolar organizer regions and proliferation index of argyrophilic nucleolar organizer regions were lower, while Bcl-2 expression was higher in group II compared with group I. The results from this study suggest that safe high-dose dexamethasone administration time is difficult to establish. Long-term, high-dose dexamethasone administration can cause pronounced morphological changes in hepatocytes by diminishing their transcriptional and proliferation activity but also protects them from apoptosis by potentially affecting Bcl-2 expression.

Expression of Proliferating Cell Nuclear Antigen (PCNA) in breast carcinoma and its association with Ki67 and tumor grade in mastectomy specimens

Background: Breast cancer ranks first among cancer in urban population of India, and 2nd most common in the rural population. Proliferative activity acts as an adjunct and gives prognostic importance in breast cancer. However, the best method for measurement of proliferative rate is controversial. The aim was to evaluate the association between PCNA labeling index (PCNA-LI) and Ki67 labeling index (Ki67-LI) and their correlation with histological grade. Methods: Seventy cases of simple and modified Radical Mastectomy specimens with the diagnosis of carcinoma breast were included in the study. H&E stained sections were examined for assessment of histological grade. IHC scoring was done for the markers Ki-67 and PCNA. Correlation of PCNA with Ki67 and tumor grade was performed. Result: 18 specimens were grade 1 tumors, 36 cases were grade 2 and 16 were grade 3. 90.0% of tumors were positive for PCNA index and Ki-67 positivity was observed in 85.7% cases, median and mean values of PCNA and Ki67 were 52.0, 51.64+/-37.475, and 24.50, 26.17± 24.183 respectively. Final score was divided into low and high index using median value as a cut off. Ki-67 LI showed strong positive correlation with histological grade (p=0.0001). PCNA LI showed positive correlation with histological grade (p=0.041) but no correlation was observed with Ki-67 LI (p=0.232) Conclusion: PCNA marker cannot substitute Ki67 but may be used as an additional marker to assess proliferative activity along with other markers. It may be useful to categorize subgroups with different grades of proliferative activity due to its easy applicability and assessment techniques. DOI: 10.21276/apalm.2017.1191

The role of atorvastatin on the restenosis process post-PTA in a diabetic rabbit model

BackgroundRestenosis remains to be a major limitation of percutaneous transluminal angioplasty (PTA) for diabetic patients with peripheral vascular disease (PVD). Despite of stations routine implements to prevent such progress, its exact effect is unclear.Methods and resultsIn our study, balloon was successfully implanted in the iliac artery of atherosclerotic rabbit. Patency of the narrowed artery was interrogated using ultrasound. Atorvastatin or vehicle was administered orally to rabbits from day 0 to day 28 after double-injury surgery. On day 7, day 14, and day 28, restenotic arteries were harvested and processed for histopathlogical analysis. Our data show that, after double-injury surgery, the intima was composed mostly by SMCs at all time course in rabbits undergoing surgery process. Significant increases in stenosis rates were noted from day 7 to day 14 (from 21 ± 5.85 % to 60.93 ± 12.46 %). On day 28 after double-injury surgery, severe restenosis was observed and daily administration of atorvastatin cannot prevent restenosis’ formation (88.69 ± 3.71 % vs. 90.02 ± 3.11 %, P > 0.05). The PCNA index and SMCs proliferation were correlated with the scores of the vascular pathology.ConclusionsOur results indicate that double-injury model can mimic clinical restenosis, based on this model, atorvastatin showed no therapeutic effect on restenosis process in diabetic rabbits after PTA.

Effect of standardized extract of Bacopa monnieri (CDRI-08) on testicular functions in adult male mice

Ethnopharmacological relevanceBacopa monnieri (BM) has been used in India since the time of Rig-Veda for augmentation of learning, memory, brain health etc. Aim of the studyThe memory augmenting effect of BM is well documented. CDRI-08 is a standardized extract of Bacopa monnieri, but its effect on the male reproductive health has not been investigated. Therefore, the aim of the present study was to examine the effect of CDRI-08 administration on the male reproductive organs with special emphasis on testis in adult mice. Materials and methodsCDRI-08, containing at least 55% bacosides (the major constituent of BM), was investigated for its effect on testicular functions in adult Parkes (P) mice. A suspension of CDRI-08 was orally administered in doses of 40 and 80mgkg−1 body weight day−1 for 28 days and various male reproductive end points were evaluated. ResultsCompared to control, CDRI-08 treatment caused a significant increase (p<0.05) in spermatogenic cell density (germinal epithelial height: control, 55.03±4.22 vs 40mg, 67.15±2.65 and 80mg, 69.93±3.76; and tubular diameter: control, 206.55±2.62 vs 80mg, 253.23±12.19), PCNA index (control, 59.85±2.09 vs 40mg, 82.17±1.56 and 80mg, 84.05±3.51) and in steroidogenic indices in the testis, and in sperm viability (control, 0.67±0.010 vs 80mg, 0.80±0.04) in cauda epididymidis of the treated mice. On the other hand, however, the same treatment caused a significant decrease (p<0.05) in abnormal sperm morphology (control, 21.72±1.06 vs 40mg, 10.63±1.50 and 80mg, 15.86±0.87) in cauda epididymidis, and in lipid peroxidation level in testis of the treated mice compared to controls. ConclusionThe results suggest that treatment with CDRI-08 extract improves sperm quality, and spermatogenic cell density and steroidogenic indices in the testis of P mice.