Journal: Viruses

Article Title: Susceptibility of HPV-18 Cancer Cells to HIV Protease Inhibitors

doi: 10.3390/v16101622

Figure Lengend Snippet: Effect of lopinavir HIV protease inhibitor on the viability of HeLa cells after 24 h treatment ( A ), 48 h treatment ( B ), and 72 h treatment ( C ). Lopinavir significantly ( p ≤ 0.01 **/ p ≤ 0.001 ***) reduced the viability of HeLa cells in a time and concentration-dependent manner. “ns” indicates a non-significant effect.

Article Snippet: The Muse ® (Merck-Millipore, Darmstadt, Germany) assay kits (Muse ® Cell Cycle Assay, Muse ® Count and Viability Assay, Muse ® Annexin V and Dead Cell Assay) and lopinavir were all purchased from Merck-Millipore (Darmstadt, Germany).

Techniques: Protease Inhibitor, Concentration Assay

Journal: Viruses

Article Title: Susceptibility of HPV-18 Cancer Cells to HIV Protease Inhibitors

doi: 10.3390/v16101622

Figure Lengend Snippet: Confirmation of percentage viability of HeLa cells in response to treatment with lopinavir ( A ), Aluvia tablets ( B ), atazanavir ( C ), and Ritoataz tablets ( D ). All the HIV drugs significantly ( p ≤ 0.001 ***) inhibited the viability of HeLa cells in vitro when compared to the untreated control. “ns” indicates a non-significant effect.

Article Snippet: The Muse ® (Merck-Millipore, Darmstadt, Germany) assay kits (Muse ® Cell Cycle Assay, Muse ® Count and Viability Assay, Muse ® Annexin V and Dead Cell Assay) and lopinavir were all purchased from Merck-Millipore (Darmstadt, Germany).

Techniques: In Vitro, Control

Journal: Viruses

Article Title: Susceptibility of HPV-18 Cancer Cells to HIV Protease Inhibitors

doi: 10.3390/v16101622

Figure Lengend Snippet: Average % apoptosis in lopinavir ( A ), Aluvia ( B ), atazanavir ( C ), and Ritoataz ( D )-treated HeLa cells. HIV protease inhibitors and curcumin (40 μM) significantly ( p ≤ 0.01 **/ p ≤ 0.001 ***) induced apoptosis cell death in HeLa cells relative to the untreated control. “ns” indicates a non-significant effect.

Article Snippet: The Muse ® (Merck-Millipore, Darmstadt, Germany) assay kits (Muse ® Cell Cycle Assay, Muse ® Count and Viability Assay, Muse ® Annexin V and Dead Cell Assay) and lopinavir were all purchased from Merck-Millipore (Darmstadt, Germany).

Techniques: Control

Journal: Viruses

Article Title: Susceptibility of HPV-18 Cancer Cells to HIV Protease Inhibitors

doi: 10.3390/v16101622

Figure Lengend Snippet: Statistical analysis of the total apoptosis induced by HIV-PIs in HeLa cells.

Article Snippet: The Muse ® (Merck-Millipore, Darmstadt, Germany) assay kits (Muse ® Cell Cycle Assay, Muse ® Count and Viability Assay, Muse ® Annexin V and Dead Cell Assay) and lopinavir were all purchased from Merck-Millipore (Darmstadt, Germany).

Techniques:

Journal: Viruses

Article Title: Susceptibility of HPV-18 Cancer Cells to HIV Protease Inhibitors

doi: 10.3390/v16101622

Figure Lengend Snippet: The effect of lopinavir ( A ), Aluvia ( B ), atazanavir ( C ) and Ritoataz ( D ) on cell cycle progression. The solvent controls had no significant effect (ns) on the progression of the cell cycle in HeLa cells when compared to the untreated control. All the HIV protease inhibitors significantly ( p ≤ 0.01 ## / p ≤ 0.001 ### ) halted the cell cycle progression at the G0/G1 phase.

Article Snippet: The Muse ® (Merck-Millipore, Darmstadt, Germany) assay kits (Muse ® Cell Cycle Assay, Muse ® Count and Viability Assay, Muse ® Annexin V and Dead Cell Assay) and lopinavir were all purchased from Merck-Millipore (Darmstadt, Germany).

Techniques: Solvent, Control

Journal: British Journal of Cancer

Article Title: HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer

doi: 10.1038/s41416-024-02774-9

Figure Lengend Snippet: TNBC cell lines MDA-MB-231 and BT549, non-TNBC cell lines MCF-7 and BT474, and TNBC-primary cells were treated for 1 h with increasing doses of bortezomib ( a ) or carfilzomib ( b ). Subsequently, the cells were placed in a drug-free medium for the next 48 h. The data represent the mean ± SD of 3 independent experiments. Cell lines were treated for 1 h with increasing doses of bortezomib ( c ) or carfilzomib ( d ). Subsequently, the cells were placed in a drug-free medium or incubated with 10 µM nelfinavir or lopinavir for the next 48 h. The data represent the mean ± SD of 3 independent experiments. Viability at the selected time point at which CDI was calculated is depicted in Fig. , . TNBC patient-derived cells were treated for 1 h with increasing doses of bortezomib ( e ) or carfilzomib ( f ). Subsequently, the cells were placed in a drug-free medium or incubated with 10 µM nelfinavir or lopinavir for the next 48 h. Cell viability was measured 48 h after treatment, and the data represent the mean ± SD of 2 independent experiments. Viability at a selected time point at which CDI was calculated is depicted in Fig. , . In all experiments, the corresponding IC 50 values were determined from the dose-response curves and are presented in Supplementary Table . g Cytotoxicity of carfilzomib and nelfinavir in the three PDxOs. Numbers represent the percentage of growth inhibition. Drug synergy was modelled using SynergyFinder+, CDI of the most synergistic drug combination is presented. BTZ bortezomib, CFZ carfilzomib, LPV lopinavir, NFV nelfinavir.

Article Snippet: The concentrations of PIs bortezomib and carfilzomib, HIV-PIs nelfinavir and lopinavir, and PgP inhibitor reserpine (Med Chem Express, Monmouth Junction, NJ, USA) are specified in the relevant sections.

Techniques: Incubation, Derivative Assay, Inhibition

Journal: British Journal of Cancer

Article Title: HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer

doi: 10.1038/s41416-024-02774-9

Figure Lengend Snippet: a BiP-GFP FRAP analysis in MDA-MB-231 cells treated with carfilzomib, lopinavir, or their combination. FRAP images were acquired 1 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. The data represent the mean ± SD of T half (in ms) recovery of BiP-GFP fluorescence in the individual cells in three independent experiments. Statistical significance was determined with one-way ANOVA with Tukey post-test. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001. b Induction of spliced XBP1 (sXBP1) presented as a ratio of spliced versus unspliced XBP1 RNA variants normalized to GAPDH and a time-point 30 min prior to the treatment. c Representative western blot image of the cleavage of ATF6 protein, represented by a cleaved form of ATF6 and obtained 2 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. GAPDH served as an internal loading control. d Induction of ATF3 expression normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. e Induction of total XBP1 (tXBP1) expression, normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. f Induction of BIP expression normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. g Induction of CHOP expression normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. h Induction of NOXA expression normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. i Representative western blot image of the induction of PARP on a protein level, represented by a cleaved form of PARP p85 and obtained 24 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. GAPDH served as an internal loading control. j Induction of early and late apoptosis represented by Anexin V + and PI− + PI+ positivity, evaluated by flow cytometry 48 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with an unpaired two-sided t-test. * represents p < 0.05, *** represents p < 0.001. In all qPCR experiments, the data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with one-way ANOVA with Tukey post-test. * represents p < 0.05, *** represents p < 0.001. ATF3 Activating Transcription Factor 6, ATF6 Activating Transcription Factor 6, BiP-GFP Binding immunoglobulin Protein green-fluorescent protein, BTZ bortezomib, CFZ carfilzomib, CHOP DNA Damage-Inducible Transcript 3, GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase, LPV lopinavir, mero-GFP Mammalian Endoplasmic Reticulum-localized redox-sensitive Green-Fluorescent Protein, NOXA Phorbol-12-Myristate-13-Acetate-Induced Protein 1, PARP Poly(ADP-Ribose) Polymerase 1, XBP1 X-Box Binding Protein 1.

Article Snippet: The concentrations of PIs bortezomib and carfilzomib, HIV-PIs nelfinavir and lopinavir, and PgP inhibitor reserpine (Med Chem Express, Monmouth Junction, NJ, USA) are specified in the relevant sections.

Techniques: Fluorescence, Western Blot, Control, Expressing, Flow Cytometry, Binding Assay

Journal: British Journal of Cancer

Article Title: HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer

doi: 10.1038/s41416-024-02774-9

Figure Lengend Snippet: a Correlation between spliced XBP1 expression level, represented as a ratio of spliced / unspliced XBP1 and normalized to GAPDH, and the sensitivity to bortezomib, represented by the IC 50 values. b Correlation between spliced XBP1 expression level, represented as a ratio of spliced/unspliced XBP1 and normalized to GAPDH, and the sensitivity to carfilzomib, represented by the IC 50 values. In both a and b , TNBC are marked in red. The data represent the mean of two independent experiments. c Representative western blot image of IRE1α knock-out in control or in two single-cell derived clones #7 and #11 of MDA-MB-231 cell line (upper part), leading to a functional decrease of sXBP1 evaluated by qPCR, assessed as a ratio of spliced vs unspliced XBP1 and normalized to GAPDH, which served as a housekeeping gene (bottom part). d Cytotoxicity of carfilzomib in MDA-MB-231 cells with a normal level of IRE1α (NC) or in single-cell derived clones (#7 and #11) with knocked-out IRE1α. Viability was assessed after 1 h pulse treatment and continuous 48 h incubation in drug-free medium. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with an unpaired t -test. e Cytotoxicity of lopinavir in MDA-MB-231 cells with a normal level of IRE1α (NC) or in single-cell derived clones (#7 and #11) with knocked-out IRE1α. Viability was assessed after 48 h continuous treatment. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with unpaired t -test. f Cytotoxicity of carfilzomib and lopinavir combination in MDA-MB-231 cells with a normal level of IRE1α (NC) or in single-cell derived clones (#7 and #11) with knocked-out IRE1α. Viability was assessed after 1 h pulse treatment with carfilzomib and continuous 48 h treatment with lopinavir. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with an unpaired t-test. BTZ bortezomib, CFZ carfilzomib, GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase, IRE1α Inositol-Requiring Enzyme 1, LPV lopinavir, XBP1 X-Box-Binding Protein 1.

Article Snippet: The concentrations of PIs bortezomib and carfilzomib, HIV-PIs nelfinavir and lopinavir, and PgP inhibitor reserpine (Med Chem Express, Monmouth Junction, NJ, USA) are specified in the relevant sections.

Techniques: Expressing, Western Blot, Knock-Out, Control, Derivative Assay, Clone Assay, Functional Assay, Incubation, Binding Assay

Journal: British Journal of Cancer

Article Title: HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer

doi: 10.1038/s41416-024-02774-9

Figure Lengend Snippet: a Representative gel image of residual activity of the proteasome β2, β5, and β1 subunits, visualized by ABP labelling 8 h after the treatment. MDA-MB-231_Ub G76V GFP cells were treated with carfilzomib alone for 1 h and subsequently placed into drug-free medium or into medium containing 10 µM lopinavir for 8 h. b Median fluorescence intensity (MFI) of Ub G76V -GFP, corresponding to functional proteasome inhibition in cells treated with carfilzomib alone or co-treated with lopinavir for 8 h. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with two-way ANOVA and Sidak’s post-test, *** represents p < 0.001. c Viability corresponding to a and b . The cells were treated with carfilzomib for 1 h and subsequently placed into a drug-free medium or into the medium with lopinavir for 48 h. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with two-way ANOVA and Sidak’s post-test, ** represents p < 0.01; *** represents p < 0.001. d Representative gel image of residual activity of the proteasome β2, β5, and β1 subunits, visualized by ABP labelling and corresponding poly-Ub accumulation 1 h after the treatment. MDA-MB-231_Ub G76V GFP cells were treated for 1 h with 10 µM lopinavir, 100 nM bortezomib, or 100 nM carfilzomib in monotherapy or in combination, as well as with β5 specific inhibitor NC005, β2 specific inhibitor LU102 or their combination. e MFI of Ub G76V -GFP, representing functional proteasome inhibition 8 h after the treatment. The cells were treated with PIs, as shown in d for 1 h and subsequently placed into a drug-free medium or into the medium with lopinavir for 8 h. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with one-way ANOVA with Dunnet post-test, *** represents p < 0.001. f Viability corresponding to d and e . The cells were treated with PIs for 1 h and subsequently placed into a drug-free medium or into a medium containing lopinavir for 48 h. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with one-way ANOVA with Dunnet post-test, *** represents p < 0.001. BTZ bortezomib, CFZ carfilzomib, GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase, LPV lopinavir, MFI median fluorescence intensity, Ub G76V -GFP mutated uncleavable ubiquitin moiety-Green Fluorescent Protein.

Article Snippet: The concentrations of PIs bortezomib and carfilzomib, HIV-PIs nelfinavir and lopinavir, and PgP inhibitor reserpine (Med Chem Express, Monmouth Junction, NJ, USA) are specified in the relevant sections.

Techniques: Activity Assay, Fluorescence, Functional Assay, Inhibition

Journal: British Journal of Cancer

Article Title: HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer

doi: 10.1038/s41416-024-02774-9

Figure Lengend Snippet: a Functional inhibition of ABCB1 (PgP) multi-drug transporters by lopinavir and reserpine in 4 studied cell lines and patient-derived primary cells. The data represent the mean MFI of MTG ± SD from 3 independent experiments for cell lines and the single value for patient-derived primary samples. Statistical significance was determined with unpaired t -test, ** represents p < 0.01; *** represents p < 0.001. b Correlation between fold change of MFI of MTG by lopinavir and fold change of IC 50 of carfilzomib by lopinavir. The data represent the correlation between the mean values from three independent experiments from cell lines and single values from patient-derived primary cells. The p -value and correlation coefficient (r) were determined by Pearson correlation. c Representative western blot image of ABCB1 level in MDA-MB-231 cells stably transduced with ABCB1 protein (PgP+). GAPDH serves as a protein loading control. d Functional inhibition of ABCB1 by lopinavir and reserpine in PgP+ and PgP- MDA-MB-231 cells. The data represent the mean MFI of MTG ± SD from 3 independent experiments. Statistical significance was determined with unpaired t-test, ** represents p < 0.01; *** represents p < 0.001. Dose-response curves of MDA-MB-231 cells without PgP (PgP-) or with introduced PgP (PgP + ) to bortezomib ( e ) and carfilzomib ( f ) in monotherapy or in combination with lopinavir. The cells were treated with PIs for 1 h and subsequently placed into a drug-free medium or into the medium with lopinavir for 48 h. The data represent the mean ± SD from 3 independent experiments. g Estimation of ABCG2 in 4 studied cell lines on the mRNA level by qPCR (upper part) and on the protein level (lower part) by western blot. In both experiments, the levels were normalized to GAPDH, which served as a control. h Representative western blot image of ABCG2 knock-out in control or in two single-cell derived clones #3 and #5 of MCF-7 cell line. i Cytotoxicity of carfilzomib in MCF-7 cells with a normal level of ABCG2 (NC) or in single-cell derived clones (#3 and #5) with knocked-out ABCG2. Viability was assessed after 1 h pulse treatment with carfilzomib and continuous 48 h incubation in a drug-free medium. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with an unpaired t-test. j Cytotoxicity of carfilzomib and lopinavir combination in MCF-7 cells with a normal level of ABCG2 (NC) or in single-cell derived clones (#3 and #5) with knocked-out ABCG2. Viability was assessed after 1 h pulse treatment with carfilzomib and continuous 48 h treatment with lopinavir. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with an unpaired t -test. ABCB1 ATP Binding Cassette Subfamily B Member 1, ABCG2 ATP Binding Cassette Subfamily G Member 2, BTZ bortezomib, CFZ carfilzomib, GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase, LPV lopinavir, MFI median fluorescence intensity, MTG MitoTracker Green FM, PgP P-glycoprotein (ABCB1).

Article Snippet: The concentrations of PIs bortezomib and carfilzomib, HIV-PIs nelfinavir and lopinavir, and PgP inhibitor reserpine (Med Chem Express, Monmouth Junction, NJ, USA) are specified in the relevant sections.

Techniques: Functional Assay, Inhibition, Derivative Assay, Western Blot, Stable Transfection, Transduction, Control, Knock-Out, Clone Assay, Incubation, Binding Assay, Fluorescence