Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: Cytotoxicity assessment of the HSF1 inhibitor NXP800 in human hepatocellular carcinoma (HCC) cell lines. ( A ) The levels of the HSF1 protein were assessed in Hep3B, HLE, HLF, Huh7, and SNU449 cell lines by Western blot analysis. GAPDH was used as a loading control. ( B ) Cell viability was measured via MTT assay. The cell viability in percentage to DMSO control, expressed as non-linear regression curve fit over log10 drug concentrations, is depicted. The mean ± SD (n = 3) of at least three independent experiments each performed in triplicate is shown. Horizontal dotted line: 50% viable cells. IC 50 values per cell line are shown in each plot.

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: Western Blot, Control, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: Effects of NXP800 on the proliferation, apoptosis, and DNA damage of HLF and SNU449 human hepatocellular carcinoma cell lines. To evaluate cell proliferation, the BrdU incorporation assay was conducted on HLF ( A ) and SNU449 ( D ) cells treated with NXP800 at concentrations of 50 nM and 500 nM for 48 h. Apoptosis was assessed in HLF ( B ) and SNU449 ( E ) cell lines after treatment with NXP800 for 48 h, using the same concentrations mentioned above. Additionally, a DNA damage assay was performed to measure the formation of apurinic/apyrimidinic (AP) sites, which represent one of the most common types of DNA lesion and a surrogate marker for DNA damage. This assay was applied to both cell lines ( C , F ) for 24 h at the two NXP800 concentrations. In all assays, untreated cells and those treated with DMSO (solvent) were used as controls. The results are presented as the mean ± standard deviation (SD) from three independent experiments, each conducted in triplicate. For statistical analysis, Tukey’s multiple comparisons test was used, with significance defined as p < 0.001. The following comparisons were conducted: a vs. untreated cells; b vs. DMSO; c vs. 50 nM NXP800.

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: BrdU Incorporation Assay, Marker, Solvent, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: Gene expression analysis in hepatocellular carcinoma cell lines treated with the HSF1 inhibitor NXP800. HLF and SNU449 cells were treated for 48 h with DMSO control (vehicle), or NXP800 (50 nM or 500 nM in HLF or SNU449 cells, respectively). Panels ( A – Q ) show the relative mRNA expression levels of the indicated genes, presented as fold change to the untreated control (Ctrl), calculated using the 2 −ΔΔCt method, with ACTB used as the housekeeping gene. Data represents the mean ± SD of three independent experiments, each measured in technical duplicates. Statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test (*** p < 0.001, **** p < 0.0001).

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: Gene Expression, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: The effect of NXP800 on glycolysis in hepatocellular carcinoma cell lines. HLF and SNU449 cells were treated with DMSO or 5 µM NXP800 for 24 h. Data are presented as mean ± SEM of triplicate samples from two independent experiments, normalized to cell number (#) as quantified by HOECHST 33342 nuclear staining. Values are multiplied by 1 × 10 5 for visualization purposes. Statistical significance compared to the DMSO control is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-way ANOVA). ( A ) The standard Glycolytic Rate Assay profile shows the basal and compensatory glycolysis over time. Cells were sequentially challenged with mitochondrial inhibitors rotenone and antimycin A (Rot/AA) to inhibit mitochondrial respiration, which helps reveal the glycolytic proton efflux rate (glycoPER). This was followed by the addition of 2-deoxy-D-glucose (2-DG) to block glycolysis and confirm the specificity of the assay. ( B , C ) The glycoPER results (left panel) and bar graphs (right panel) display key parameters such as basal glycolysis, compensatory glycolysis, basal proton efflux rate (PER), and post-2-DG acidification for ( B ) HLF and ( C ) SNU449 cells.

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: Staining, Control, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: The effect of NXP800 on mitochondrial respiration in hepatocellular carcinoma cell lines. HLF and SNU449 cells were treated with DMSO or 5 µM NXP800 for 24 h. Data are presented as mean ± SEM of triplicate samples from two independent experi-ments, normalized to cell number as quantified by HOECHST 33342 nuclear staining. Values are multi-plied by 1 × 10 5 for visualization purposes. Statistical significance compared to the DMSO control is indicated as * p < 0.05, *** p < 0.001, **** p < 0.0001 (two-way ANOVA). ( A ) The standard Cell Mitochondrial Stress Test profile shows the oxidative consumption rate (OCR) over time. Basal respiration was followed by sequential injections of oligomycin (which measures ATP-linked OCR), carbonyl cyanide-4-phenylhydrazone (FCCP; which assesses maximal respiration and spare capacity), and Rot/AA (to determine non-mitochondrial OCR). ( B , C ) The OCR profile (left panel) and bar graphs (right panel) illustrate basal respiration, maximal respiration, spare respiratory capacity, ATP production, and proton leak for ( B ) HLF and ( C ) SNU449 cells.

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: Effect of treatment with NXP800 either alone or associated with doxorubicin administration on cell proliferation, apoptosis, and DNA of HCC cell lines. To evaluate cell proliferation, the BrdU incorporation assay was conducted on HLF ( A ) and SNU449 ( D ) cells treated with NXP800 at a 50 nM concentration, either alone or in association with 50 μM doxorubicin. Apoptosis was assessed in HLF ( B ) and SNU449 ( E ) cell lines after treatment with NXP800 and doxorubicin for 24 h, using the same concentrations mentioned above. Additionally, a DNA damage assay was performed to measure the formation of apurinic/apyrimidinic (AP) sites, which represent one of the most common types of DNA lesion. This assay was applied to both cell lines ( C , F ) for 24 h at with NXP800 at a 50 nM concentration, either alone or associated with 50 μM doxorubicin. In all assays, untreated cells and those treated with DMSO (solvent) were used as controls. Treatments were limited to 24 h because massive cell death was observed at 48 h in the HCC cells subjected to the combinatorial treatment. The results are presented as the mean ± standard deviation (SD) from three independent experiments, each conducted in triplicate. For statistical analysis, Tukey’s multiple comparisons test was used, with significance defined as p < 0.001. The following comparisons were conducted: a vs. untreated cells; b vs. DMSO; c vs. 50 nM NXP800; d vs. doxorubicin.

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: BrdU Incorporation Assay, Concentration Assay, Solvent, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: Synergy analysis of NXP800 and doxorubicin in HLF and SNU449 cells. HLF and SNU449 cells were treated with increasing concentrations of NXP800 and doxorubicin as single agents and in combination in a dose–response matrix. Cell viability was assessed by MTT assay after 48 h of treatment, and drug interaction analysis was performed using the ZIP model implemented in SynergyFinder 3. ( A , E ) MTT viability assay showing the dose-dependent effect of doxorubicin monotherapy in HLF ( A ) and SNU449 ( E ) cells. ( B , F ) Dose–response curves of NXP800 and doxorubicin monotherapies generated using SynergyFinder 3. ( C , G ) Inhibition matrices displaying percent growth inhibition as a heatmap (red indicates stronger inhibition). ( D , H ) Two-dimensional ZIP synergy score maps for all tested drug combinations. Scores > 5 indicate synergistic interactions.

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: MTT Assay, MTT Viability Assay, Generated, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: The Novel HSF1 Inhibitor NXP800 Exhibits Robust Antitumor Activity in Hepatocellular Carcinoma

doi: 10.3390/ijms27062781

Figure Lengend Snippet: Effect of treatment with NXP800 either alone or associated with the PARP inhibitor olaparib (OLA) on cell proliferation, apoptosis, and DNA of HCC cell lines. To evaluate cell proliferation, the BrdU incorporation assay was conducted on HLF ( A ) and SNU449 ( D ) cells treated with NXP800 at 50 nM concentration, either alone or in association with 5 OLA. Apoptosis was assessed in HLF ( B ) and SNU449 ( E ) cell lines after treatment with NXP800 and OLA, using the same concentrations mentioned above. Additionally, a DNA damage assay was performed to measure the formation of apurinic/apyrimidinic (AP) sites. This assay was applied to both cell lines ( C , F ) for 48 h with NXP800 at 50 nM concentration, either alone or associated with 5 μM OLA. Untreated cells and those treated with DMSO (solvent) were used as controls. The results are presented as the mean ± standard deviation (SD) from three independent experiments, each conducted in triplicate. For statistical analysis, Tukey’s multiple comparisons test was used, with significance defined as p < 0.001. The following comparisons were performed: a vs. untreated cells; b vs. DMSO; c vs. 50 nM NXP800; d vs. OLA.

Article Snippet: The human hepatocellular carcinoma cell lines HLE (XenoTech, Kansas City, KS, USA; Cat. No. JCRB0404), HLF (Tebubio GmbH, Le Perray en Yvelines, France; Cat. No. JCRB0405), PLC/PRF/5 (CLS Cell Lines Service GmbH, Eppelheim, Germany; Cat. No. 300315), Huh7 (Cytion GmbH, Heidelberg, Germany; Cat. No. 300156), and SNU449 (LGC Standards GmbH, Teddington, Middlesex, UK; Cat. No. CRL-2234) were purchased as indicated and cultured according to standard procedures.

Techniques: BrdU Incorporation Assay, Concentration Assay, Solvent, Standard Deviation