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Abstract
Controlled production of cyclin dependent kinases (CDK) and stabilization of tumor suppressor genes are the most important factors involved in preventing carcinogenesis. The present study aimed to explore the cyclin dependent apoptotic effect of nymphayol on breast cancer MCF-7 cells. In our previous study, we isolated the crystal from a chloroform extract of Nymphaea stellata flower petals and it was confirmed as nymphayol (17-(hexan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol) using x-ray diffraction (XRD), Fourier transform infrared (FTIR), and mass spectroscopy (MS) methods. The cytotoxic effect of nymphayol on MCF-7 cells were analyzed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cellular and nuclear damage was determined using propidium iodide (PI) and acridine orange/ethidium bromide (AO/ErBr) staining. Tumor suppressor and apoptosis related mRNA transcript levels were determined using real-time polymerase chain reaction (RT-PCR). Nymphayol potentially inhibits MCF-7 cell viability up to 78%, and the IC50 value was observed as 2.8 µM in 24 h and 1.4 µM in 48 h. Treatment with nymphayol significantly increased reactive oxygen species (ROS) level and the tunnel assay confirmed DNA damage. We found characteristically 76% apoptotic cells and 9% necrotic cells in PI and AO/ErBr staining after 48 h treatment with 2.8 µM of nymphayol. Gene expression analysis confirmed significantly (p ≤ 0.001) increased mRNA levels of cyclin dependent kinase inhibitor 2A (Cdkn2a), retinoblastoma protein 2 (pRb2), p53, nuclear factor erythroid 2-factor 2 (Nrf2), caspase-3, and decreased B-cell lymphoma 2 (Bcl-2), murine double minute 2 (mdm2), and proliferating cell nuclear antigen (PCNA) expression after 48 h. Nymphayol effectively inhibited breast cancer cell viability, and is associated with early expression of Cdkn2a, pRb2, and activation of p53 and caspases.
Highlights
Incidence of cancer are recognized with altered apoptosis mechanism, genetic mutations, oxidative stress, hypoxia, and sustained intra cellular inflammation, while environmental factors are linked to ultraviolet ray exposure, radiation, and lifestyle [1]
The Nymphaea stellata chloroform extract yielded a total of 74 fractions; each fraction was spotted on a precoated Silica gel 60 F254, 0.25 mm thick thin layer chromatography (TLC) plate (Merck) and eluted in a hexane:ethyl acetate (4:1) system and fractions with similar Rf values in TLC pattern were pooled together
We found that nymphayol increased reactive oxygen species (ROS) generation and stimulated hyper proliferative signals in MCF-7 cells, which effectively increased the early expression of cyclin dependent kinase (CDKN2A), aiding in the accumulation of active tumor suppressor p53
Summary
Incidence of cancer are recognized with altered apoptosis mechanism, genetic mutations, oxidative stress, hypoxia, and sustained intra cellular inflammation, while environmental factors are linked to ultraviolet ray exposure, radiation, and lifestyle [1]. Aberrant cellular mechanisms in the apoptotic signaling pathway results in uncontrolled cell progression, leading to carcinogenesis [2]. Stimulating or suppressing apoptotic factors such as tumor suppressor genes and cyclin dependent kinases aid in arresting cancer progression. The tumor suppressor p53 is stabilized and accumulated during uncontrolled cancer progression, but in stress or hyper proliferation signaling conditions, mdm negatively regulates p53, which leads to the transition from resting phase (G1) to DNA synthesis phase (S), and subsequently cancer cell progression continues. The cell-cycle gatekeeper gene p14ARF neutralizing mdm function via cyclin dependent protein kinase (Cdkn2A) leads to increased levels of active p53 [5]. Targeting or early activation of CDKs and retinoblastoma (RB) resulting in the activation of p53 tumor-suppressor pathways is a fundamental requirement for the genesis of most human cancer treatment [6]
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