Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: Visualization of the morphology of MG63 and APR1 osteosarcoma cells by epifluorescence microscopy under control (DMSO-treated) and regorafenib-treated (IC50) conditions. Key cellular structures were visualized via fluorescent dyes: the nuclei were stained with DAPI (blue), the actin cytoskeleton was stained with phalloidin Atto-488 (green), and the mitochondrial network was stained with mitoRed (red). Magnification 100x, scale bar: 200 µm; 200x, scale bar: 100 µm.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Epifluorescence Microscopy, Control, Staining

Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: Representative images of the wound healing assay captured for MG63 ( a ) and for APR1 ( b ), along with wound gap width measurements ( c ). The analysis assessed the impact of regorafenib at the IC50 concentration on the invasive and proliferative potential of MG63 and APR1 cell lines measured at starting point (0 hour) and after 24 hours. Images were taken at 100-fold magnification; scale bar: 400 µm. Statistical significance was indicated with asterisks ****p<0.0001. Data are presented as mean ± SD from n = 3 independent biological replicates, with two technical replicates per condition and two images analyzed per each replicate using ImageJ.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Wound Healing Assay, Concentration Assay

Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: The invasion of the osteosarcoma cell lines MG63 and APR1 is modulated by regorafenib. Representative images ( a ) and quantitative analysis ( b ) illustrating the invasive capacity of MG63 and APR1 cells following treatment with regorafenib at the IC50 concentration. Magnification: 40-fold and 100-fold; scale bar: 100 µm. Statistical significance was indicated via asterisks ****p<0.0001. Data are shown as mean ± SD based on three independent biological replicates, each assessed in two technical replicates, with two images per replicate subjected to ImageJ analysis. Results are expressed as the percentage of migrated cells per field and normalized to the corresponding control conditions.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Concentration Assay, Control

Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: Apoptosis profiles were determined by flow cytometry. Regorafenib at the IC50 decreased the number of viable MG63 and APR1 cells ( b ) while simultaneously increasing the percentage of necrotic ( c ), early apoptotic ( d ), and late apoptotic ( e ) cells. On the basis of the dot plots ( a ), four distinct cell populations were identified: viable cells (Annexin V and PI negative), early apoptotic cells [EA] (Annexin V positive, PI negative), late apoptotic cells [LA] (Annexin V and PI positive), and dead (necrotic) cells (Annexin V negative, PI positive). Statistically significant differences are indicated by asterisks (**p < 0.01 and ****p < 0.0001). Data are presented as mean ± SD from n = 2 biological replicates, each performed in 3 technical replicates.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Flow Cytometry

Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: Influence of regorafenib IC50 treatment on the mRNA and protein levels of BAX ( a and b ), BCL-2 ( c and d ), and MCL-1 ( h and i ) in MG63 and APR1 cell lines. Additionally, the BAX/BCL-2 ratio was determined for both mRNA and protein expression ( e and f ). Gene expression was quantified via RT‒qPCR, with normalization to a reference gene, and transcript levels were calculated via the RQmax algorithm. For RT-qPCR experiments, analyses were performed using two independent biological replicates, with each biological sample analyzed in three technical replicates. Representative Western blot images are presented ( g ), and the corresponding protein levels were quantified through densitometric analysis. Western blot analysis consisted of two independent biological replicates, each performed with two technical replicates. Statistical significance was indicated using asterisks as follows: *p < 0.05, ***p<0.001, ****p<0.0001. Differences that were not statistically significant are marked as “ns”.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Western Blot

Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: The DNA content distribution in the osteosarcoma cell lines was assessed via flow cytometry. Representative histograms ( a ) illustrate the proliferative status of cells across the cell cycle phases in the control and regorafenib-treated groups. The cells were classified into three distinct populations: G0/G1 phase, S phase, and G2/M phase ( b ). Statistically significant differences are indicated by asterisks (**p < 0.01 and ***p < 0.001), whereas comparisons with no statistically significant differences are marked as “ns”. Values represent the mean ± SD derived from two independent biological replicates, each including three technical replicates.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Flow Cytometry, Control, Derivative Assay

Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: Effect of regorafenib treatment at the IC 5 0 concentration on noncoding RNA expression in MG63 and APR1 cell lines. The expression levels were quantified by RT-qPCR, normalized to a reference gene, and calculated via the RQ Max algorithm. The graphs show the expression of selected microRNAs: miR-17-5p ( a ), miR-21-5p ( b ), miR-140-5p ( c ), miR-155-5p ( d ) and long noncoding RNAs: lncMALAT ( e ), lncTUG ( f ). Statistically significant differences are indicated by asterisks (**p < 0.01 and ****p < 0.0001). Data represent the mean ± standard deviation of two independent biological experiments, each performed in three technical replicates.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Concentration Assay, RNA Expression, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: Effect of regorafenib at the IC 5 0 concentration on the expression profile of genes associated with cancer-related pathways in MG63 ( a ) and APR1 ( b ) osteosarcoma cell lines. The x-axis of volcano plot represents log 2 (fold regulation) and the y-axis −log 1 0 (p-value). Upregulated genes are marked with red upward arrows, whereas downregulated genes are marked with green downward arrows. Genes consistently up- or downregulated in both MG-63 and APR-1 cells are additionally highlighted with light-red or light-green circles, respectively. Vertical dashed lines indicate the fold-change cutoff used to define differential expression.Genes showing expression changes of at least 2-fold were classified as differentially expressed. Gene expression levels were normalized to those of reference genes ( ACTB , B2M , RPLP0 , and GAPDH ) and calculated using the 2^(-ΔΔCt) method. The reactions for this assay were performed using three independent biological replicates, each processed as one technical replicate.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Concentration Assay, Expressing, Quantitative Proteomics, Gene Expression

Journal: Cancer Management and Research

Article Title: Regorafenib-Induced Stress Response Alters the Bioenergetic Profile of Osteosarcoma Cells and Modulates Gene Expression Associated with Metabolic regulation-a Potential Mechanism of Osteosarcoma Treatment-Related Adaptation

doi: 10.2147/CMAR.S562346

Figure Lengend Snippet: Heatmap illustrating the expression profiles of all 84 genes involved in pathways associated with oncogenesis. The heatmap provides a comprehensive overview of gene expression modulation in MG63 and APR1 osteosarcoma cell lines following treatment with regorafenib at the IC50 concentration.

Article Snippet: Regorafenib was purchased from MedChemExpress (New Jersey, USA) and dissolved in sterile DMSO (Sigma Aldrich/Merck, Poznań, Poland) to a final concentration of 1 mM.

Techniques: Expressing, Gene Expression, Concentration Assay