Jacqueline J. Stevens

Previous in vitro studies in our laboratory demonstrated that ethyl acetate (P₂) and water- soluble (PS/PT1) fractionated leaf extracts of Ocimum gratissimum inhibit the proliferation of prostate cancer cells. It has been reported that the crude aqueous extract induces apoptosis in lung adenocarcinoma cells; however, the efficacy of the fractionated extracts against these cells remains unclear. In the present study, we hypothesized that the ability of the fractionated extracts to inhibit proliferation and induce apoptosis is associated with the activation of pro-apoptotic proteins and induction of DNA condensation in A549 cells. Ocimum gratissimum was cultivated and its leaves were harvested, extracted, and fractionated to produce fractions P₂ and PS/PT1. Anti-proliferative activity was assessed by direct cell count. For morphological characterization of apoptosis, 4′,6-diamidino-2-phenylindole staining was employed. Western blot analysis was performed to evaluate the apoptotic activity of the fractionated extracts. In data generated from anti-proliferation studies, P₂ significantly inhibited cell proliferation in a concentration-dependent manner; PS/PT1 elicited a decrease in the viability of cells, occurring at 500 µg/mL. 4′,6-diamidino-2-phenylindole staining revealed the induction of apoptosis, as evidenced by the formation of apoptotic bodies. Increased levels of pro-apoptotic proteins were observed as the concentrations of the fractionated extracts increased. These results suggest that fractionated leaf extracts of Ocimum gratissimum inhibit the proliferation and induce apoptosis of A549 cells.

Funded by the National Institutes of Health (NIH), the Research Centers in Minority Institutions (RCMI) Program fosters the development and implementation of innovative research aimed at improving minority health and reducing or eliminating health disparities. Currently, there are 21 RCMI Specialized (U54) Centers that share the same framework, comprising four required core components, namely the Administrative, Research Infrastructure, Investigator Development, and Community Engagement Cores. The Research Infrastructure Core (RIC) is fundamentally important for biomedical and health disparities research as a critical function domain. This paper aims to assess the research resources and services provided and evaluate the best practices in research resources management and networking across the RCMI Consortium. We conducted a REDCap-based survey and collected responses from 57 RIC Directors and Co-Directors from 98 core leaders. Our findings indicated that the RIC facilities across the 21 RCMI Centers provide access to major research equipment and are managed by experienced faculty and staff who provide expert consultative and technical services. However, several impediments to RIC facilities operation and management have been identified, and these are currently being addressed through implementation of cost-effective strategies and best practices of laboratory management and operation.

Colon cancer is the third leading cause of cancer-related deaths worldwide. Recent studies in our laboratory have demonstrated that arsenic trioxide is cytotoxic in human colon cancer (HT-29), lung (A549) and breast (MCF-7) carcinoma cells. The purpose of the present study is to investigate the effects of arsenic trioxide on DNA synthesis and the possible genotoxic effects on human colon cancer cells. HT-29 cells were cultured according to standard protocol, followed by exposure to various doses (0, 2, 4, 6, 8, 10, and 12 μg/mL) of arsenic trioxide for 24 h. The proliferative response (DNA synthesis) to arsenic trioxide was assessed by [³H]thymidine incorporation. The genotoxic effects of arsenic-induced DNA damage in a human colon cancer cell line was evaluated by the alkaline single cell gel electrophoresis. Results indicated that arsenic trioxide affected DNA synthesis in HT-29 cells in a biphasic manner; showing a slight but not significant increase in cell proliferation at lower levels of exposure (2, 4 and 6 µg/mL) followed by a significant inhibition of cell proliferation at higher doses (i.e., 8 and 10 µg/mL). The study also confirmed that arsenic trioxide exposure caused genotoxicity as revealed by the significant increase in DNA damage, comet tail-lengths, and tail moment when compared to non-exposed cells. Results of the [³H]thymidine incorporation assay and comet assay revealed that exposure to arsenic trioxide affected DNA synthesis and exhibited genotoxic effects in human colon cancer cells.

Arsenic trioxide, the trade name Trisenox, is a drug used to treat acute promyleocytic leukemia (APL). Studies have demonstrated that arsenic trioxide slows cancer cells growth. Although arsenic influences numerous signal-transduction pathways, cell-cycle progression, and/or apoptosis, its apoptotic mechanisms are complex and not entirely delineated. The primary objective of this research was to evaluate the effects of arsenic trioxide on DNA synthesis and to determine whether arsenic-induced apoptosis is mediated via caspase activation, p38 mitogen–activated protein kinase (MAPK), and cell cycle arrest. To achieve this goal, lung cancer cells (A549) were exposed to various concentrations (0, 2, 4, 6, 8, and 10 µg/mL) of arsenic trioxide for 48 h. The effect of arsenic trioxide on DNA synthesis was determined by the [3H]thymidine incorporation assay. Apoptosis was determined by the caspase-3 fluorescein isothiocyanate (FITC) assay, p38 MAP kinase activity was determined by an immunoblot assay, and cell-cycle analysis was evaluated by the propidium iodide assay. The [³H]thymidine-incorporation assay revealed a dose-related cytotoxic response at high levels of exposure. Furthermore, arsenic trioxide modulated caspase 3 activity and induced p38 MAP kinase activation in A549 cells. However, cell-cycle studies showed no statistically significant differences in DNA content at subG1 check point between control and arsenic trioxide treated cells.

Arsenic is a heavy metal that exhibits a high degree of toxicity to various organ systems. In humans, this compound is associated with an increase risk of skin cancer, and may cause cancers of the lung, liver, bladder, kidney, and colon. The mechanism of arsenic-related carcinogenicity remains to be elucidated. Hence, the aim of the present study was to investigate the cytotoxic effects of arsenic trioxide (As2O3) on adenocarcinoma colorectal cancer (HT-29) cells using the MTT [3-(4,5 dimethylthiazoyl-2-yl)-2,5- diphenyltetrazolium bromide] assay for cell viability. To achieve this objective, HT-29 cells were cultured and exposed to various doses (0, 2, 4, 6, 8, 10, 12, and 14 μg/ml) of arsenic trioxide for 24 h, 48 h, and 72 h respectively, and subsequently assessed for viability following a standard MTT test protocol. Experimental data indicated that arsenic trioxide is cytotoxic to colon cancer cells showing LD50 values of 9.8, 9.4 and 9.0 μg/ml upon 24, 48 and 72 h of exposure, respectively. There was a dose-dependent response with regard to As2O3 toxicity in HT-29 cells. Although there was a reduction in LD50 value with increasing exposure time, this decrease was not statistically significant.

Ultraviolet (UV)-induced cataracts are becoming a major environmental health concern because of the possible decrease in the stratospheric ozone layer. Experiments were designed to isolate gene(s) affected by UV irradiation in rabbit cornea tissues using fluorescent differential display-reverse transcription-polymerase chain reaction (FDDRT-PCR). The epithelial cells were grown in standard medium for 2 or 4 hours post treatment. Cornea epithelial cells were irradiated with UVB for 20 minutes. RNA was extracted and amplified by reverse transcriptase-polymerase chain reaction using poly A⁺ specific anchoring primers and random arbitrary primers. Polyacrylamide gel electrophoresis revealed several differentially expressed genes in untreated versus UV irradiated cells. Complimentary DNA (cDNA) fragments resulting from fluorescent differentially expressed mRNAs were eluted from the gel and re-amplified. The re-amplified PCR products were cloned directly into the PCR-TRAP cloning system. These data showed that FDDRT-PCR is a useful technique to elucidate UV-regulated gene expressions. Future experiments will involve sequence analysis of cloned inserts. The identification of these genes through sequence analysis could lead to a better understanding of cataract formation via DNA damage and mechanisms of prevention.