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Comparative proteomic analysis of side and non-side populations isolated from A549 lung carcinoma cells cultivated under normoxic and hypoxic conditions

ABSTRACT: Presence of key hypoxic regulators, HIF-1α or HIF-2α, in tumors is associated with disease progression and poor patient prognosis. Hypoxia massively activates several genes including the one encoding BCRP transport protein that proffers the multidrug resistance to cancer cells through the xenobiotic efflux and, moreover, is a determinant of side population (SP) that associates with cancer stem-like phenotype. As the natural medicine comes to the fore, it is instinctive to look for natural agents possessing powerful features against cancer resistance. Hypericin, pleiotropic agent found in Hypericum plants, is good example, as it is BCRP substrate and potential inhibitor, as well as SP- and HIF-modulator. Here, we showed that hypericin efficiently accumulates in hypoxic cancer cells, degrades HIF-1/2α and together with hypoxia decreases BCRP efflux, thus diminishes SP population. On the contrary, this seemingly favorable result was accompanied by stimulated migration of this minor population of cancer cells that preserved SP phenotype. Since hypoxia unexpectedly decreased BCRP level and SP fraction, we also compared the SP and nonSP proteomes and their changes under hypoxia in A549 cell line. We identified differences especially among protein groups connected to the epithelial-mesenchymal transition, although major changes were related to hypoxia, as upregulation of many proteins, including serpin E1, PLOD2 and LOXL2, that ultimately contribute to the initiation of the metastatic cascade, were detected. Altogether, this study represents another piece of puzzle that clarify the innate and hypoxia-triggered resistance of cancer cells and highlights the ambivalent role of natural agents in biology of these cells.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Pavel Roudnický

LAB HEAD: Zbyněk Zdráhal

PROVIDER: PXD038740 | Pride | 2023-05-04

REPOSITORIES: Pride

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			Action	DRS
	3899_Vargova_150min-IS_top20_IT50_HCD_10_HB-3.raw	Raw
	3899_Vargova_150min-IS_top20_IT50_HCD_11_NA-3.raw	Raw
	3899_Vargova_150min-IS_top20_IT50_HCD_12_NB-3.raw	Raw
	3899_Vargova_150min-IS_top20_IT50_HCD_13_HA-4.raw	Raw
	3899_Vargova_150min-IS_top20_IT50_HCD_14_HB-4.raw	Raw

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Project description:Melanoma remains the most lethal skin cancer, mainly because of high resistance to therapy. Side population (SP) cells are found in many types of cancer and are usually enriched in therapy-resistant as well as tumorigenic cells. Here, we identified a Hoechst dye-effluxing SP in a large series of human melanoma samples representing different progression phases. The SP size did not change with disease stage but was correlated with the prognostic “Breslow’s depth” in the primary (cutaneous) tumors. When injected into immunodeficient mice, the SP generated larger tumors than the bulk “main population” (MP) melanoma cells in two consecutive generations, and showed tumorigenic capacity at lower cell numbers than the MP. In addition, the SP reconstituted the heterogeneous composition of the human A375 melanoma cell line, and its clonogenic activity was 2.5-fold higher than that of the MP. Gene-expression analysis revealed upregulated expression in the melanoma SP (versus the MP) of genes associated with chemoresistance and anti-apoptosis. Consistent with these molecular characteristics, the SP increased in proportion when A375 cells were exposed to the melanoma standard chemotherapeutic agent dacarbazine, and to the aggravating condition of hypoxia. In addition, the SP showed enhanced expression of genes related to cell invasion and migration, as well as to putative (melanoma) cancer stem cells (CSC) including ABCB1 and JARID1B. ABCB1 immunoreactivity was detected in a number of tumor cells in human melanomas, and in particular in clusters at the invasive front of the primary tumors. Together, our findings support that the human melanoma SP is enriched in tumorigenic and chemoresistant capacity, considered key characteristics of CSC. The melanoma SP may therefore represent an interesting therapeutic target. Four primary melanomas (subtype SSMM) were dissociated into single cells. From each of these single cell suspensions, side population (SP) and bulk \main population\ (MP) cells were isolated based on their Hoechst-effluxing capability using fluorescence-activated cell sorting. Gene expression of SP cells is analyzed relative to the gene expression of the corresponding MP cells.

Project description:The activation of the transcription factor Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small-molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high throughput screen led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations but did not affect expression levels of genes outside of the HIF-1 pathway. Lead structure BAY 87-2243 was found to inhibit HIF-1Î± protein accumulation under hypoxic conditions in NSCLC cell line H460 but had no effect on HIF-1Î± protein accumulation and HIF target gene expression in RCC4 cells lacking VHL activity or in H460 cells after inhibition of HIF prolyl hydroxylase activity. BAY 87-2243 had no effect on HIF-Î±-mRNA levels. Antitumor activity of BAY 87-2243 and suppression of HIF-1 target gene expression in vivo was demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial production of reactive oxygen species (ROS) by blocking complex I activity but has no effect on complex III activity. Lowering of mitochondrial ROS production to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors. We used microarrays to detail the global programme of gene expression that is induced in NSCLC cell line H460 upon hypoxia (16 h incubation at 1 % pO2) and evaluated a dose-dependent effect of our HIF-1-pathway inhibitor BAY 87-2243 on genes tthat are affected by hypoxia. Specificity of BAY 87-2243 for the suppression of HIF-1-mediated gene transcription on a genome-wide scale was evaluated by microarray hybridizations using Affymetrix GeneChip Human Gene 1.0 ST arrays. RNA from normoxic H460 cells and from hypoxic H460 cells incubated with 1, 10 and 100 nM BAY 87-2243 respectively was subjected to array hybridization. Of those 30 genes that were most strongly suppressed by 100 nM BAY 87-2243 in hypoxic H460 cells compared to DMSO-treated hypoxic H460 cells, virtually all of them are induced by prior hypoxia and most of these genes have been described in the literature as HIF-1 target genes

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Comparative proteomic analysis of side and non-side populations isolated from A549 lung carcinoma cells cultivated under normoxic and hypoxic conditions

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