| BackgroundProstate cancer is the second most recurrently diagnosed cancer and sixth leading cause of cancer death in males, worldwide. Traditional chemotherapy and radiotherapy have not proven to provide significant survival benefits to patients with advanced prostate cancer, where most available treatment options are palliative only. There has raised an urgent action for search of new molecules to combat the rising cases of prostate cancer, especially for those androgen-independent prostate cancer Afzelin is a flavonol glycoside found in Nymphaeaodorata. It has been found to inhibit the growth of breast cancer cells by stimulating apoptosis. It has also been found to scavenge superoxide anion radical in RAW264.7 cells and it also inhibits cyclooxygenase (COX)-1, COX-2 and lipid peroxidation. The structure of this compound is shown in Figure 1.The crucial biological functions such as the provision of a structural frame work and driving force for cellular function and division are mediated by the actin cytoskeleton in all eukaryotic cells. Tumor cell metastasis is a multi-step process driven by dynamic reorganization of the actomyosin cytoskeleton and remodelling of the extracellular matrix, allows cells to cross tissue boundaries and spread via blood and lymphatic vessels to distal regions of the body. A comprehensive overview of the biological processes that regulate the organization of actin is of recent interest to the regime of cancer therapy.Members of the Rho GTPase family such as myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKa) are key regulators of the actin cytoskeleton and together with multiple target proteins safe guard the tight regulation of normal cell growth and differentiation. In the event of carcinogenesis or genetic alterations, eukaryotic cells becomes predisposed to uncontrollable and rapid growth, which is evidenced by the elevated levels of LIMK1 kinase, a kinase which itself is activated by MRCKa, ROCK1 (Rho-associated, coiled-coil-containing protein kinase 1) and ROCK2 which are expressed in prostate cancer. ROCK inhibitors have been shown to reduce the invasive ability of tumor cells in vitro and to prevent the in vivo dissemination of tumor cells including breast cancer, fibrosarcoma, liver, lung, melanoma and also prostate cancer. The inhibitors of these biochemical entities are thought to restore normal cell proliferation and provide a key solution in cancer treatment. Thus the current study was aimed to evaluate in-vitro anti prostate cancer activity of afzelin and its effect on prostate cancer related kinases.ObjectiveThe aim of this study was to evaluate in vitro anti prostate cancer activity of Afzelin and its effect on prostate cancer related kinases.Methods Cell CultureAndrogen-sensitive LNCaP (Lymph Node Carcinoma of the Prostate) and androgen-independent PC-3 (Prostate Cancer-3) prostate cancer cell lines were obtained (American Type Culture Collection, Manassas, VA) and the cells were maintained in EMEM supplemented with 10% fetal bovine serum, glutamine, and 2% penicillin-streptomycin, and 0.2% gentamicin. PC-3 cells were grown in Ham’s F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (90%) plus 10% fetal bovine serum, as recommended by American Type Culture Collection. The cells were maintained at 37℃ in a humidified atmosphere of 95% air and 5% CO2.All chemicals used in this study were of analytical grade and were obtained from Sigma-Aldrich, (St.Louis, MO, USA) unless otherwise specified.Cell proliferation assayFor determining the cell proliferation, cells (LNCaP and PC-3) were seeded in 96-well plates at a density of 5000 cells/well with a final volume of 100μL. After seeding for 24 h, the medium was removed, replaced with fresh medium or with medium containing afzelin at a concentration of 0.1,1.0 and 10.0 ug/mL in a final volume of 100 μL. The culture was maintained in the CO2 incubator (Bio-Equip, Shanghai Yiheng Technology Co., Ltd, China) for an additional period of 3 days at 37℃ in a humidified atmosphere of 95% air and 5% CO2. For each afzelin concentration (0.1,1.0 and 10.0 ug/mL) and time point, cultures were maintained in quadruplicates. At the end of the 24,48 and 72 h time periods,10 μL of WST-1 labelling solution (WST-1 cell proliferation assay kit; Roche Diagnostics, Indianapolis, IN) was added and the cells were returned to the incubator for a period of 2 h. The absorbance of the formed formazan product was detected at 420 nm in a 96-well spectrophotometric plate reader, as described by the manufacturer (DiaSorin Inc, Stillwater, MN, USA).Cell cycle analysisFor determination of critical cell cycle and the regulatory proteins, cultured prostate cancer cells (LNCaP and PC-3) were exposed to afzelin (0.1,1.0 and 10.0 ug/mL) for 24 h and adherent cells were trypsinized and pooled with the cells in suspension and washed three times with ice-cold PBS (phosphate buffered saline). A fraction of these washed cells were stained with trypan blue and counted to determine cell viability. The cells were adjusted to 1×106 cells/mL and fixed in a 2:1 ratio (v/v) in chilled methanol overnight before staining with propidium iodide in the presence of RNase. Cell cycle distribution was analyzed on a Becton Dickinson (San Jose, CA) flow cytometer, and at least 10,000 cells were analyzed for each experimental condition. Data analysis was performed using CellQuest cell cycle analysis software (version 5.1).Western blotting analysisFor determining the effects of afzelin on the expression of proteins (MRCKa, LIMK1 and ROCK1 kinases), both LNCaP and PC-3 prostate cancer cells were treated with varying concentrations of afzelin (0.1,1.0 and 10.0 μg/mL). The protein concentrations was measured spectrophotometrically using a modified Lowry assay protocol (DC Protein Assay; Bioroad, Hercules, CA). Equivalent amounts of proteins were separated on SDS-polyacrylamide gel (7.5% -12%) and blotted to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were incubated with blocking solution (PBS containing 0.05% Tween 20 and 5% nonfat dry milk) to block non-specific binding and incubated with primary antibodies followed by corresponding horse-radish peroxidise conjugated secondary antibodies [anti MRCKa, LIMK1 and anti ROCK1 antibodies (Catlog#R10-63R) raised in rabbit manufactured by SignalChem, Canda was used according to manufacturer’s instructions]. The positive bands were visualized by using enhanced chemiluminescence solution (GE Healthcare).ResultsAfzelin decreases the in vitro growth of prostate cancer cellsThis study has analyzed the effect of afzelin on the cell growth of two prostate cancer cell lines (LNCaP and PC-3). The cells were exposed to increasing concentrations of afzelin (0.1,1.0 and 10.0 μg/mL) for 24 h after seeding and their viability was followed for 72 h using the WST-1 cell proliferation assay. Cell growth over the 72 h time period for both prostate cancer cell lines treated with afzelin is shown in Fig.2 A and B respectively. The dose response curves are presented as percentage of control growth on the corresponding day, and each value was measured in quadruplicates. The figures indicate that an exposure to afzelin at concentrations 1.0 μg/mL and 10.0 μg/mL strongly inhibited cell growth for both cell lines. There was no growth inhibition evident in the vehicle (0.1% DMSO)-treated control cells. Afzelin was found to be a more potent inhibitor of cell growth in both LNCaP and PC-3 cells.Afzelin induces apoptosis in both LNCaP and PC-3 cellsFlow cytometric analysis was used to examine the cell cycle distribution of both the LNCaP and PC-3 cells in the absence and presence of afzelin for 24 h. As seen in Fig.3A, a broad peak of cells equalling 27.6% of the total cell population appeared in GO population of the afzelin treated (10.0 μg/mL) PC-3 cells, as compared with only 12.7% of the control. Also,43.3% of the cells accumulated in G1 phase of cell cycle. This elevated the PC-3 cells localizing in the GO region. Interestingly, same kind of results were obtained in LNCaP cells where LNCaP cells showed 12.0% of the total cell population localised in GO phase as compared to 19.4% in afzelin treated (10.0 μg/mL) LNCaP cells (Fig.3B). Here also 50.7% cells got accumulated in G1 phase. This elevated population of cells localizing in the GO peak after afzelin treatment is suggestive of an ongoing extensive apoptotic process or cytotoxic response. The obtained results suggest that afzelin has selective actions on both cell types.Afzelin decreased the expression of members of the Rho GTPase familyproteinsTo investigate molecular effects of afzelin on these prostate cancer cell lines, we conducted a focused analysis to determine how afzelin treatment might influence the expression of some critical molecules involved in cell cycle progression and apoptosis. Western blot analysis of the effects of increasing concentrations of afzelin on members of the Rho GTPase family proteins like LIMK1, MRCKa and ROCK1 expression depicted that phosphorylation of these kinases progressively diminished in both LNCaP and PC-3 cells as the dose was increased (Fig.4A). However, suppression was more significant in PC-3 cells as showed by densitometry evaluation (Fig.4B)..ConclusionAfzelin,with these promising results can prove to be a potent candidate in cancer therapy in prostate cancer. |
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