Antiproliferative activity of marine stingray Dasyatis sephenvenom on human cervical carcinoma cell line

AbstractBackgroundVenoms comprise mixtures of numerous bioactive compounds that have a wide range of pharmacologic actions. Toxins from venomous animals have attracted the attention of researchers because of their affinity for primary sites responsible for lethality and their efficacy at extremely low concentrations. The venoms of marine stingrays have not been extensively studied and limited data is available on them. The present study aims to evaluate the antiproliferative and biochemical properties of the venom obtained from a species of marine stingray (Dasyatis sephen) on human cervical cancer cell line HeLa.MethodsThe antiproliferative effect of D. sephen venom was determined by MTT assay, and the oxidative stress was determined by lipid peroxidation method along with assessment of changes in the enzymatic and non-enzymatic antioxidant status. We observed intracellular reactive oxygen species (ROS) levels by DCFH-DA method, mitochondrial membrane potential alterations by rhodamine 123 staining and apoptotic morphological changes by acridine orange/ethidium bromide dual staining method.ResultsD. sephen venom enhances lipid peroxidative markers such as thiobarbituric acid reactive substance, conjugated diene, and lipid hydroperoxide in HeLa cell lines. Stingray venom enhances the ROS levels, which is evidenced by the increased 2–7-diacetyl dichlorofluorescein fluorescence. Further, D. sephen venom treatment altered the mitochondrial membrane potential in HeLa cells. Additionally, we observed increased apoptotic morphological changes in D. sephen venom-treated groups. ConclusionsDasyatis sephen venom exhibits potent antiproliferative effect on HeLa cell line and upon further purification it could be a promising antiproliferative agent

Additional file 1: of Antiproliferative activity of marine stingray Dasyatis sephen venom on human cervical carcinoma cell line

Scientific classification and image of D. sephen. (DOCX 105 kb

Fibroblast-derived ECM decreases Lung Cancer Cell Growth.

<p>(A) A549, H358 and HPL1D cells were grown on fibroblast derived matrices and every day one well of cells was trypsinized and manually counted in triplicate using trypan blue. (B) A549, H358 and HPL1D cells were grown on fibroblast derived matrices and every day Alamar Blue was added to wells of cells in triplicate and relative conversion of Alamar Blue was determined. n = 3. *, p-value ≤ 0.05.</p

Fibroblast-derived ECM alter Lung Cancer Cell Line Morphology.

<p>(A) Phase contrast microscopy photos of A549, H358, and HPL1D cells on FN, WI38 ECM, IMR90, and HDF ECM. (B) Immunofluorescent confocal microscopy of A549 cells on WI38-derived ECM stained with Phallodin and DAPI. (C) The circularity of A549 on FN and 3 different fibroblast-derived ECM was calculated. n = 10. *, p ≤ 0.05.</p

Fibroblast-derived ECM alters mRNA profile of A549 and H358 Cells.

<p>Heat map significantly changed genes from microarray (fold-change >1.5 and p-value ≤ 0.05). Columns represent individual gene probes; rows are the different samples and each row represents one of the biological triplicates ran on the microarray. (B) Validation of microarray data. Representative genes were chosen for quantitative real-time qRT-PCR analysis. New biological triplicates were prepared, RNA extracted and converted to cDNA, and real-time qRT-PCR was performed. All samples tested were validated and all genes were changed in the same direction as the microarray, however some amplitudes of change were slightly different.</p

Fibroblast-derived ECM alters Protein Levels of A549 Cells.

<p>(A) A549 cells were cultured on the indicated substrate for 48 hours and then cells were harvested and western blots were performed with the indicated antibodies. (B) Cells were treated as in panel A and westerns were performed with indicated antibodies. (C) A549 cells were seeded on either fibronectin (FN) or WI38-derived ECM and then plates were incubated under hypoxic or normoxic conditions. Cells were harvested and westerns were performed with the indicated antibodies.</p

Fibroblast-derived ECM Protects Lung Cancer Cell Lines from Serum Deprivation.

<p>(A) A549, H358, and HPL1D cells were grown on fibronectin (FN) or on WI38 ECM in serum-free media for 48 hours and then photographed. (B) Relative cell numbers were quantified for A549 cells (B), H358 cells (C) and HPL1D cells (D) using Alamar Blue every 24 hours after cells were put into serum free media. By the fourth day in serum free media, basically all cells were dead.</p

Characterization of fibroblasts and their ECM.

<p>A. Microscopic analysis of fibroblasts and de-cellularized matrix. Left colum- 5X phase contrast imaging of confluent fibroblasts just prior to de-cellularazation. Middle colum- 40X phase contrast microscopy of ECM following decellularization. Right column- Immunofluorescent confocal microscopy of fibroblast-derived ECM using an NHS-ester probe conjugated to Alexa Fluor 488. (B) Protein content of ECM was quantitated after scraping of the ECM from decellularized plates. Protein amount is indicated a μg/cm², N = 3. (C) SDS-PAGE and colloidal blue stain of fibroblast-derived ECM. n = 3.</p