Fig 1.
Pancreatic cancer cell proliferation is decreased upon M344 treatment.
After treatment with M344 at various concentrations, the proliferation of (A) S2-013, (B) BxPC-3, (C) MIA PaCa-2, (D) T3M-4, and (E) CFPAC-1 cells was assessed by the MTT assay at 24, 48, and 72 hours. The graphs display the optical density (OD) at 570 nm relative to the OD for the 0.1% DMSO control. Each error bar represents the standard error of the mean. These results are representative of findings in 2–3 separate experiments for each cell line. The results from 0.1% DMSO control treatment versus treatment with each M344 concentration were compared using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 2.
Pancreatic cancer cell viability is diminished by M344 treatment.
After treatment with M344 at various concentrations, the viability of (A) S2-013, (B) BxPC-3, (C) MIA PaCa-2, (D) T3M-4, and (E) CFPAC-1 cells was assessed by the trypan blue exclusion assay at 24, 48, and 72 h. Viability was graphed as the number of live cells/total cells x 100. Each error bar represents the standard error of the mean. These results are representative of findings in 2–3 separate experiments for each cell line. The results from 0.1% DMSO control treatment versus treatment with each M344 concentration were compared using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the following p values: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 3.
In pancreatic cancer cells, M344 causes cell cycle arrest in G1.
Treatment of S2-013 cells with 1 or 10 μM M344 resulted in large increases in the populations accumulated in G1 at 24 hours (A), 48 hours (B), and 72 hours (C), as shown by propidium iodide staining and flow cytometry. Statistical comparisons were made using a Two-way ANOVA with Tukey’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the following p values: * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001.
Fig 4.
M344-induced apoptosis is apparent by 48 hours and necrosis peaks at 72 hours.
S2-013 cells were treated with 0.1% DMSO control or M344 (1 μM or 10 μM) for 24, 48, or 72 hours. Caspase-3 and caspase-7 cleavage was simultaneously analyzed by using the CellEventTM Caspase-3/7 Green Flow Cytometry Assay Kit. The SYTOXTM AADvancedTM Dead Cell Stain included in the kit identified necrotic cells. Each error bar represents the standard error of the mean. Statistical comparisons of the results were done using a Two-way ANOVA with Tukey’s multiple comparisons test. The asterisks indicate the following p values: * <0.05, ** <0.01, *** <0.001, **** <0.0001.
Fig 5.
Migration of pancreatic cancer cells is reduced upon treatment with M344.
Transwell assays were performed to assess the migration of (A) S2-013, (B) BxPC-3, (C) MIA PaCa-2, (D) T3M-4, and (E) CFPAC-1 cells following treatment with 1 μM or 10 μM of M344 (or with 0.1% DMSO, as the vehicle control) for 24 h. For the transwell assays, after treatment the cells were plated into 3 separate 8-μm inserts (1x105 cells/insert) in the presence of 1 μM M344, 10 μM M344, or 0.1% DMSO and incubated for 24 h. Cells were fixed and stained, and photographs of 3 random fields were taken for each of the inserts. The results of counts of the stained, migrated cells were averaged. The graph shows the average numbers of cells that migrated, and each error bar represents the standard error of the mean. The presented graph for the S2-013 cells is representative of the results from 3 separate experiments; the full experimental set of counts for each of the other cell lines was done once. The results of 0.1% DMSO control treatment versus the 1 μM or the 10 μM M344 treatment were compared using Student’s t test. The asterisks indicate the following p values: * p<0.05, ** p< 0.01, *** p<0.001, and **** p<0.0001.
Fig 6.
Compared to vorinostat, M344 decreases pancreatic cancer cell proliferation more effectively for a longer duration.
The proliferation of S2-013 cells was assessed by the MTT assay following treatment with 0.1% DMSO control or with 1 μM, 5 μM, 10 μM, or 25 μM M344 or vorinostat for 48 hours or 72 hours. The effects of M344 versus vorinostat at 48 hours (A) and at 72 hours (B) are shown. The same data are displayed for M344 (C) and vorinostat (D) at 48 hours versus 72 hours. The error bars represent the standard error of the mean. The results were compared using a Two-way ANOVA with Tukey’s multiple comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the following p values: ** p< 0.01, *** p<0.001, **** p<0.0001.
Fig 7.
M344 treatment of pancreatic cancer cells increases global histone H3 acetylation more than vorinostat treatment.
The inhibition of histone H3 deacetylation in S2-013 cells by M344 versus vorinostat was compared by monitoring the percentage of acetylated histone H3 following 48-hour treatment with M344 or vorinostat (at 1 μM or 10 μM) or with the 0.1% DMSO vehicle alone. Global H3 acetylation was assessed using the EpiQuikTM Global Histone H3 Acetylation Assay Kit. The data are displayed in (A) for 1 μM M344 and vorinostat (and 0.1% DMSO control), and in (B) for 10 μM M344 and vorinostat (and 0.1% DMSO control). These data were compiled from two biological replicates with triplicate samples in the first assay and quintuplet samples in the second assay. The graphs display the percentage global histone H3 acetylation, calculated by this formula: OD (treated sample–blank) / OD (untreated control–blank) X 100%. Error bars represent the standard error of the mean. Statistical comparisons were made with One-Way ANOVA with Tukey’s multiple comparisons tests. The asterisks indicate the p values: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 8.
M344 does not impair HEK293 cell viability more than vorinostat.
After treatment with M344 or vorinostat at the indicated concentrations, the viability of HEK293 cells was assessed by the trypan blue exclusion assay at 24, 48, or 72 hours. Viability was graphed as the number of live cells/total cells x 100. Each error bar represents the standard error of the mean. The results from the 0.1% DMSO control treatment versus treatment with each M344 and vorinostat concentration were compared using a Two-way ANOVA with Sidak’s multiple comparisons test. The asterisks indicate the p value: * p<0.05.
Fig 9.
M344 treatment increases the expression of the human MHC class I heavy chains HLA-B and -C.
For immunoblot analysis, lysates of S2-013 pancreatic cancer cells treated for 24 or 48 hours with 0.1% DMSO (vehicle control) or with 1, 5, 10, or 25 μM M344 were subjected to electrophoresis, and the proteins were transferred to a membrane that was cut and one section was probed with an antibody for HSC70 (as a loading control) and another section was probed with an antibody to detect the MHC class I heavy chain (specifically, with the HC10 antibody that binds to HLA-B and HLA-C heavy chains). The exposure for the HLA-B/C heavy chains that is shown in the figure is longer than the exposure for the HSC70 control. Similar results were also obtained using the same M344 concentrations, time points, and antibodies in a separate experiment using different S2-013 lysates.
Fig 10.
MHC class I expression on S2-013 pancreatic cancer cells is increased following M344 treatment.
After 24- or 48-hour treatments with 0.1% DMSO (vehicle control) or with M344 at 5 or 10 μM concentrations, cell surface expression was monitored using flow cytometry with the BB7.2 antibody for peptide-occupied HLA-A2 and the B1.23.2 antibody that detects HLA-B/C. The bar graphs depict the median fluorescence intensity (MFI) fold change relative to the 0.1% DMSO control treatment for (A) 24-hour M344 treatment and (B) 48-hour M344 treatment. Triplicate wells for each concentration and time point were analyzed. Each error bar represents the standard error of the mean. Statistical comparison of the results from the 0.1% DMSO control treatment versus treatment with each M344 concentration was performed using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons Test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001. Representative histogram overlays are shown for S2-013 (C) HLA-A2 expression at 24 hours post-treatment with M344, (D) HLA-A2 at 48 hours post-treatment with M344, (E) HLA-B/C at 24 hours post-treatment with M344, and (F) HLA-B/C at 48 hours post-treatment with M344. In the histograms, solid lines represent 0.1% DMSO treatment, dashed lines represent 5 μM M344 treatment, and solid gray areas represent 10 μM M344 treatment.
Fig 11.
S2-013 pancreatic cancer cell proliferation is decreased by treatment with the combination of M344 and gemcitabine more than by either treatment alone at 48 or 72 hours post-treatment.
The proliferation of S2-013 cells following treatment with DMSO, 10 μM M344, 100 nM gemcitabine, or 10 μM M344 + 100 nM gemcitabine was monitored by MTT assay at the indicated time points. These results are representative of the data from 4 separate experiments. The error bars represent the standard error of the mean. The results were compared using Ordinary One-way ANOVA with Tukey’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * p<0.05, ** p< 0.01, *** p<0.001, **** p<0.0001.
Fig 12.
M344 impairs viability in combination with gemcitabine.
S2-013 pancreatic cancer cells were treated with 0.1% DMSO, 10 μM M344, 100 nM gemcitabine, or 10 μM M344 + 100 nM gemcitabine. Viability was assessed by the trypan blue exclusion assay at 24, 48, and 72 hours and graphed as the number of live cells/total cells x 100. Each error bar represents the standard error of the mean. The results from 0.1% DMSO control treatment versus treatment with each M344 concentration were compared using Ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2. The asterisks indicate the p values: * p<0.05, ** p< 0.01, **** p<0.0001.
Fig 13.
M344 decreases orthotopic pancreatic tumor growth when used as a treatment alone or in combination with gemcitabine.
(A) S2-013 cells were orthotopically implanted into the pancreas of female NU/J mice. After 8 days, the tumor volume for each mouse was monitored twice weekly with the VisualSonic Vevo 3100 Imaging System. At 15 days post-implantation of tumor cells, the mice were randomized into control or treatment groups with matched average tumor volumes. M344 was administered intraperitoneally at 10 mg/kg for 5 days per week (5 days on, 2 days off). Gemcitabine was given every 3 days intraperitoneally at 50 mg/kg. On Day 25 post-tumor implantation, the mice were euthanized and the tumors were resected and weighed. The changes in tumor volume over time are shown in (B) and representative images of tumors at 25 days post implantation are shown in (C). For statistical analysis, ordinary One-way ANOVA with Dunnett’s Multiple Comparisons test in GraphPad Prism Version 8.4.2 was used. The asterisks indicate the following p values: * p<0.05, ** p< 0.01, *** p<0.001.