Proteome changes in human bladder T24 cells induced by hydroquinone derived from Arctostaphylos uva-ursi herbal preparation.

ETHNOPHARMACOLOGICAL RELEVANCE: Arctostaphylos uva-ursi (L.) Spreng. (bearberry) is a well-known traditional herbal plant used as a urinary tract disinfectant. Its antiseptic and diuretic properties can be attributed to hydroquinone, obtained by hydrolysis of arbutin. AIM OF THE STUDY: This study aimed to determine the toxic profile of free hydroquinone on urinary bladder cells (T24) as a target of therapeutic action. MATERIALS AND METHODS: Quantitative and qualitative analysis of the extract and the digestive stability and bioavailability of arbutin and hydroquinone were performed by HPLC assay and simulated in vitro digestion, respectively. Cytotoxic effect, reactive oxygen species induction and proteome changes in T24 cells after hydroquinone treatment were determined using Neutral red assay, 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay and mass spectrometry, respectively. RESULTS: Through in vitro digestion, arbutin was stable, but hydroquinone increased after pepsin treatment (109.6%) and then decreased after the small intestine phase (65.38%). The recommended doses of Uva-ursi had a cytotoxic effect on T24 cells only when all hydroquinone conjugates were converted to free hydroquinone (320 and 900 µg/mL) and the toxic effect was enhanced by recovery. One cup of the therapeutic dose had a prooxidative effect after 4 h of incubation. Shorter time of cell exposure (2 h) to hydroquinone did not have any impact on reactive oxygen species induction. Proteomic analysis found 17 significantly up-regulated proteins compared to control. Hydroquinone activated proteins related to oxidative stress response, stress-adaptive signalling, heat shock response and initiation of translation. CONCLUSIONS: Despite the therapeutic properties of bearberry, up-regulated T24 cell proteins are evidence that plant compounds, although from a natural source, may exhibit negative properties.

Adenovirally mediated p53 overexpression diversely influence the cell cycle of HEp-2 and CAL 27 cell lines upon cisplatin and methotrexate treatment.

PURPOSE: p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. METHODS: p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. RESULTS: In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. CONCLUSIONS: This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and on the chemotherapeutics' modality of action.

Atypical cytostatic mechanism of N-1-sulfonylcytosine derivatives determined by in vitro screening and computational analysis.

We have previously shown that N-1-sulfonylpyrimidine derivatives have strong antiproliferative activity on human tumor cell lines, whereby 1-(p-toluenesulfonyl)cytosine showed good selectivity with regard to normal cells and was easily synthesized on a large scale. In the present work we have used an interdisciplinary approach to elucidate the compounds' mechanistic class. An augmented number of cell lines (11) has allowed a computational search for compounds with similar activity profiles and/or mechanistic class by integrating our data with the comprehensive DTP-NCI database. We applied supervised machine learning methodology (Random Forest classifier), which offers information complementary to unsupervised algorithms commonly used for analysis of cytostatic activity profiles, such as self-organizing maps. The computational results taken together with cell cycle perturbation and apoptosis analysis of the cell lines point to an unusual mechanism of cytostatic action, possibly a combination of nucleic acid antimetabolite activity and a novel molecular mechanism.

The novel C-5 aryl, alkenyl, and alkynyl substituted uracil derivatives of L-ascorbic acid: synthesis, cytostatic, and antiviral activity evaluations.

The novel C-5 substituted uracil derivatives of l-ascorbic acid were synthesized by coupling of 5-iodouracil-4,5-didehydro-5,6-dideoxy-l-ascorbic acid with unsaturated stannanes under Stille reaction conditions. The new compounds were evaluated for their antitumoral and antiviral activities. Among all compounds evaluated the 5-propynyl substituted uracil derivative of l-ascorbic acid (7) exhibited the most pronounced cytostatic activities against all examined tumor cell lines (IC(50): 0.2-0.78 microM). However, this compound was also cytotoxic to human normal fibroblasts WI 38. The 5-(phenylethynyl)uracil-2,3-di-O-benzylated l-ascorbic acid derivative (4) exhibited an albeit slight (IC(50): 55-108 microM), but selective inhibitory effect toward all tumor cell lines except for cervical carcinoma (HeLa), pancreatic carcinoma (MiaPaCa-2), laryngeal carcinoma (Hep-2), and colon carcinoma (SW 620), and no cytotoxicity to normal human fibroblast (WI 38). Compound 7 showed some, not highly specific, inhibitory potential against vesicular stomatitis virus, Coxsackie B4 virus, and Sindbis viruses (EC(50): 1.6 microM).