Abstract
Purpose: The hypoxia in solid tumors is associated with the resistance to chemo/radiotherapy. Hypoxia-inducible factor-1 (HIF-1) plays a key role in cell remodeling to hypoxia. Therefore, the inhibition of HIF-1 accumulation is considered a hopeful strategy for the treatment of cancer. Here, we aimed to evaluate the geno- and cytotoxicity properties of sclareol, a natural bicyclic diterpene alcohol, on A549 cells in CoCl2-induced hypoxia. Methods: The cytotoxicity and apoptosis-inducing properties of sclareol on the A549 cell were evaluated using MTT assay and Annexin V/PI staining, respectively in hypoxia. DAPI staining, DNA ladder, and comet assay were used to evaluate the genotoxicity. Further, the qPCR technique was employed to assess the expression of HIF-1α, HIF-1β, and downstream target genes (GluT1, and Eno1). Finally, the level of HIF-1α protein was evaluated through Western blotting in sclareol-treated cells in hypoxia. Results: The inhibitory concentration (IC50) of sclareol against A549 cells was 8 μg/mL at 48 hours in hypoxia. The genotoxicity of sclareol was confirmed in the cells treated with sclareol in hypoxia. Sclareol induced ~46% apoptosis and also necrosis in the hypoxic condition. The qPCR analyses showed an enhanced suppression of HIF-1α, HIF-1β, GluT1, and Eno1 due to the sclareol treatment in the hypoxia. Moreover, protein quantification analysis showed dose-dependently degradation of HIF-1α in hypoxia upon treatment with sclareol. Conclusion: The results obtained here indicate that sclareol possesses dose-dependent cytotoxicity effects against A549 cells in hypoxia through inhibition of HIF-1α protein accumulation, increasing cell sensitivity to intracellular oxygen levels, and disruption of cell adaptation to hypoxia.
Highlights
The unlimited proliferation of tumor cells results in a deficiency in nutrition and oxygen.[1]
Hypoxia-inducible factor-1 (HIF-1) acts as an oxygen sensor in which is regulated through oxygen-dependent approaches[3] and oxygen-independent pathways (e.g., PI3K, AKT, and MAPK).[4]
Various investigations showed that the expression of different proteins involved in metastasis and premetastasis of cancer cells is associated with the levels of nucleus HIF-1.5 The HIF-1 transcription factor is a family of basic–helix-loop-helix and composed of HIF-1α and HIF-1β subunits.[6]
Summary
The unlimited proliferation of tumor cells results in a deficiency in nutrition and oxygen.[1]. 1|Page changes and neovascularization.[2] Hypoxia-inducible factor-1 (HIF-1) acts as an oxygen sensor in which is regulated through oxygen-dependent approaches[3] and oxygen-independent pathways (e.g., PI3K, AKT, and MAPK).[4] Various investigations showed that the expression of different proteins involved in metastasis and premetastasis of cancer cells is associated with the levels of nucleus HIF-1.5 The HIF-1 transcription factor is a family of basic–helix-loop-helix (bHLH) and composed of HIF-1α and HIF-1β subunits.[6] The HIF-1α level is controlled by the oxygen concentration.[7] In the normoxia (10-21% oxygen tension), the asparagine 803 in the HIF-1α subunit is hydroxylated leading to the degradation of HIF-1α by the 26S proteasome degradation system In hypoxia, it moves to the nucleus, and binds to the HIF-1β, forming a HIF-1 transcription factor.[8] An autoregulatory feedback mechanism was recommended for HIF-1α degradation in normoxia through HIF-1α t proteasome targeting factor (HPTF).
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