The anti-tubulin compound 3b and R8-PNA-a221 were synthesized by the research groups of the author RR (University of Ferrara) and Professor Roberto Corradini (University of Parma), respectively; the procedure for the synthesis of both molecules has been previously reported (32,44). For all cell cultures, RPMI-1640 medium supplemented with 10% FBS and 100 mg/ml streptomycin and 100 IU/ml penicillin was employed. RPMI-1640 medium (cat. no. BE12-702F) and PBS (cat. no. BE17-516F) were purchased from Lonza Biosciences, trypsin-EDTA solution (cat. no. 59428C) and 50,000 IU/ml streptomycin and 50 mg/ml penicillin (cat. no. 11074440001) were from Sigma-Aldrich; Merck KGaA and FBS (cat. no. S1400) was obtained from Biowest. For flow cytometric assays, the Muse^® Annexin V & Dead Cell kit (cat. no. MCH100105), Muse^® Caspase-3/7 kit (cat. no. MCH100108) and the Muse^® Cell Cycle kit (cat. no. MCH100106) were purchased from Luminex Corporation. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder (cat. no. M5655) for MTT assay, crystal violet staining solution (cat. no. V5265) for clonogenic assay, DMSO (cat. no. D2650) used to resuspend compound 3b and the TRI Reagent (cat. no T9424) used for RNA extraction were purchased from Sigma-Aldrich; Merck KGaA, while methanol (cat. no. 309001) was supplied by CARLO ERBA Reagents.

The human glioma U251 (51) and T98G (49) cells were employed in the present study; both cell lines were purchased from Sigma-Aldrich; Merck KGaA. For anti-proliferative assay using the U251 cell line, 8×10⁴ cells were seeded in a 12-well plate, and after 4 h the cells were treated with a serial dilution of the test compounds (compound 3b was used at the 1, 2, 4, 6 and 8 µM concentrations and R8-PNA-a221 at the 1, 2, 4 and 8 µM concentrations). The cells were incubated for 72 h at 37°C in a humidified 5% CO₂ atmosphere. Following 72 h of incubation, cells were detached from the plate by trypsinization and counted using a BECKMAN COULTER^® Z2 cell counter (Beckman Coulter, Inc.). The IC50 value (50% inhibitory concentration) is defined as the concentration of compound that inhibits cell proliferation by 50% (44). The IC50 values presented (± standard deviation) are the average values derived from three independent experiments.

Following each treatment, the cells were observed and representative images were acquired using a Nikon Eclipse 80i microscope (Nikon Corporation), in order to observe whether any morphological changes occurred in the cells following treatment.

For determining the cytotoxic effects of the test compounds, MTT assay was performed using the U251 cell line (50). Cells were seeded in a 96-multi-well plate at a density of 8×10³ cells/well and after 4 h, treated with the lower concentrations of compound 3b (4 µM) and R8-PNA-a221 (2 µM) individually and in combination. Cells were incubated at 37°C for a further 72 h and at the end of the incubation period, MTT was added to each well at a final concentration of 0.5 mg/ml. Following 3 h of incubation at 37°C, the medium was discarded, and dimethyl sulfoxide (DMSO) was added; the plate was stirred for 30 min to fully dissolve the formazan crystals formed at the bottom of the wells. The absorbance was measured at 570 nm using the SUNRISE microplate reader (Tecan Group, Ltd.).

Cells were seeded into 6-well plates (400 cells per well) in RPMI-1640 medium with 10% FBS and incubated at 37°C for 24 h prior to treatment. The plates were incubated at 37°C for a further 10 days undisturbed in the incubator. Each well was then washed twice with PBS and covered with a methanol containing fixation/staining solution that allows the simultaneous coloration and fixation of the cells (crystal violet aqueous solution 0.5%/methanol, 1:1 ratio) for 15 min at room temperature and washed four times with tap water; the plate was air-dried for 1 day prior to obtaining images. Images were acquired using a Nikon SMZ1000 stereo zoom microscope (Nikon Corporation). Stained colonies consisting of >50 cells were manually counted, and the number was recorded (52).

Cells were detached by trypsinization (cat. no. 59428C; Sigma-Aldrich; Merck KGaA), collected by centrifugation at 1,200 rpm (8 min at room temperature), and lysed with Tri-Reagent (Sigma-Aldrich; Merck KGaA) according to manufacturer's instructions. The isolated RNA was washed once with cold 75% ethanol and stored at −80°C until use. The obtained RNA was dried and dissolved in nuclease-free water prior to use (32).

The miRNA levels were assayed using the TaqMan MicroRNA Reverse Transcription kit (cat. no. 43-665-96, Applied Biosystems; Thermo Fisher Scientific, Inc.) with RT-qPCR and miRNA-specific primers and probes (listed in Table I) obtained from Thermo Fisher Scientific, Inc.. All samples were run in duplicate using TaqMan Universal PCR Master Mix, no AmpErase UNG 2X (cat. no. 4324018; Thermo Fisher Scientific, Inc.) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.).

For PCR reactions, the following protocol was employed: 95°C for 10 min, 95°C for 15 sec, followed by a step at 60°C for 1 min (last two steps repeated for 50 cycles). Data were collected and analyzed using Bio-Rad CFX Manager Software (Bio-Rad Laboratories, Inc.). Relative gene expression was calculated using the 2^−ΔΔCq method and data normalization was performed using snRNA U6 and hsa-let-7c as reference (53).

The cells were treated with the lower and higher concentrations of compound 3b (4 and 6 µM) and R8-PNA-a221 (2 and 4 µM) individually and in combination at the lower concentration of both. Following 72 h of incubation at 37°C, the cells were detached by trypsinization, washed once in PBS and fixed with 70% EtOH for 24 h. For analysis 5×10⁵ cells were washed in PBS and resuspended in 200 µl of Muse^® Cell Cycle Reagent and incubated for 30 min at room temperature protected from light. Finally, the cell suspension was transferred into a new 1.5 ml tube without cap and the samples were analyzed by flow cytometry using Guava^® Muse^® Cell Analyzer (Luminex Corp.) (44).

Apoptosis assays were performed with the Guava^® Muse^® Cell Analyzer instrument, and its relative kits according to the instructions supplied by the manufacturer. Muse^® Annexin V & Dead Cell Kit utilizes Annexin V to detect Phosphatidyl Serine (PS), a common apoptotic marker that is exposed out of the external membrane of apoptotic cells, and 7-ADD (7-aminoactinomycin D) a DNA intercalating molecule used as an indicator of cell membrane integrity (it can bind DNA only in cells undergoing late apoptosis/death stage, when the membrane integrity is lost). Following 72 h of treatment with compound 3b (4 and 6 µM) and R8-PNA-a221 (2 and 4 µM) administered individually and the combination of the lower concentration of both, the cells were washed with sterile PBS, trypsinized and resuspended in RPMI medium supplemented with 10% FBS. Finally, 50 µl of cell suspension were incubated with 100 µl Muse^® Annexin V & Dead Cell reagent at room temperature and protected from light for 20 min. Samples were then analyzed using the Guava^® Muse^® Cell Analyzer (Luminex Corp.) and data acquired utilizing the Annexin V and Dead Cell Software Module (Luminex Corp.) (54).

For the analysis of caspase-3/7 activity following treatments, the Muse^® Caspase-3/7 kit was employed, which is based on a DNA binding dye linked to a DEVD peptide substrate. When pro-apoptotic caspase-3/7 are activated, they cleave the dye and the binding of the dye to DNA results in high fluorescence signal. This kit also contains a fluorescent DNA intercalator (7-ADD) as indicator of cell membrane integrity. Following the same treatments indicated in the Cell apoptosis assay, trypsinization was performed and 50 µl of cell suspension were incubated with 5 µl of caspase-3/7 reagent for 30 min (under strict protection from light). Following 25 min of incubation at 37°C, 150 µl of 7-AAD working solution were added to each tube and incubated for 5 min at room temperature before reading the samples. The samples were then analyzed using Guava^® Muse^® Cell Analyzer (Luminex Corp.) and data acquired utilizing the Caspase-3/7 Software Module (Luminex Corp.) (54).

Results are expressed as the mean ± standard error of the mean (SEM). Comparisons between groups were made by using ordinary one-way ANOVA followed by a post hoc multiple comparison tests. Dunnett's test was used to compare groups against a single control and Tukey's test was used to make comparisons against more than one group. P<0.05 was considered to indicate a statistically significant difference.

The chemical structures of the compounds used in the present study are presented in Fig. 1 [tetrahydrothieno[2,3-c]pyridine 3b (Fig. 1A) and PNA-a221 targeting miR-221 (Fig. 1C); these have been shown to exert a potent anti-apoptotic effect (34). Among the possible apoptotic-associated mRNAs, ATG10, CDKN1B/p27, BMF, APAF-1, PTEN, p27(kip1), p57(kip2) and PUMA have been validated as miR-221 targets (55-62). The example of PUMA mRNA is presented in Fig. 1B, exhibiting a functional miR-221 binding site in its 3′UTR sequence (61,62).

The effects of compound 3b (Fig. 2A) and R8-PNA-a221 (Fig. 2B) on the proliferation of U251 cells were examined. Following 72 h of cell culture in the indicated experimental conditions, the cell number/ml was determined. The data indicated that the inhibitory effect of compound 3b on U251 cell growth reached maximum values when 4-8 µM concentrations of compound 3b were employed. The anti-proliferative effects of R8-PNA-a221 were lower and detectable in the same range of concentrations, reaching 50% inhibition of cell growth only at the 8 µM concentration. In order to obtain preliminary information on the combinatory effect of the two drugs, two different concentrations for 3b (4 and 6 µM) and R8-PNA-a221 (2 and 4 µM) were selected.

As shown in Fig. 3, tetrahydrothieno[2,3-c]pyridines 3b did not affect the miR-221-3p content. This experiment was conducted by exposing, for 72 h, human glioma U251 cells to the indicated concentrations of compound 3b and R8-PNA-a221 and R8-PNA-a221-MUT; the mutated PNA contains 4 mismatches in the sequence (Fig. 1) which prevents it from hybridizing with the target mir-221-3p sequence, demonstrating the enhanced sensitivity of R8-PNA-a221 (32). Following this period of cell culture, RNA was isolated and miR-221-3p sequences were quantified by RT-qPCR. As was expected, the R8-PNA-a221 (but not the mutated R8-PNA-a221-MUT) inhibited the production of miR-221-3p. On the contrary, no inhibitory effects were observed with compound 3b treatment at 4 µM.

The anti-proliferative and cytotoxic effects of single and combined treatments are shown in Fig. 4. The anti-proliferative effects were assayed by employing a cell counter (Fig. 4A, left panel), while cytotoxicity was evaluated by MTT assay (Fig. 4A, right panel). A representative image of the clonogenic assay is presented in Fig. 4B. The results obtained clearly indicated that co-treatment with compound 3b and R8-PNA-a221 was associated with the lowest number of colonies, and the highest anti-proliferative and cytotoxic effects.

A morphological analysis was also performed (Fig. 4C) demonstrating that, in agreement with the enhanced effects on cell growth and vitality, the alterations of the morphology of the U251 glioma cells occurred following co-treatment with compound 3b and R8-PNA-a221. In particular the combined use of the two compounds led to major morphology alterations, including the loss of the original 'astrocytic' shape, and typical hallmarks of apoptosis, such as membrane blebbing.

Since the mechanisms of action of anti-tubulin compounds, such as compound 3b involved causing cell cycle alterations, the effects of compound 3b and PNA-a221 on the cell cycle were investigated by flow cytometric analysis in the present study. The cells were treated and incubated for 72 h as indicated in the experiment illustrated in Fig. 5A. Compound 3b induced a marked increase in the number of cells in the G2/M phase of the cell cycle, associated with a decrease in the number of cells in the G0/G1 phase. As shown in Fig. 5A, treatment of the U251 cells with compound 3b at 4 and 6 µM increased the percentage of cells in the G2/M phase from 12.3% (control group) to 46.9 and 47.6%, respectively. This finding is in agreement with data obtained using human K562 leukemia cells previously reported (44) and confirms that compound 3b affects cell growth through cell cycle blockade. On the contrary, the R8-PNA-a221 exerted only minor effects on cell growth, inducing a very low increase in the percentage of cells in the G2/M phase from 12.3% (control group) to 16.7-19.1%. The minimal effect of 8-PNA-a221 on the cell cycle was confirmed by the fact that combined treatment with 4 µM compound 3b and 2 µM R8-PNA-a221 induced a cell cycle distribution very similar to that induced by treatment with compound 3b alone. The quantitative data of the effects of the treatments on the cell cycle are presented in Fig. 5B. When the results of FACS analyses shown in Fig. 5A were considered, it was evident that an increase in the sub-G1 cell population occurred when the cells were treated with compound 3b, R8-PNA-a221 or the combination of compound 3b and R8-PNA-a221. As shown in Fig. 5C, a marked increase in the number of cells in the sub-G1 phase was observed when the U251 cells were treated with compound 3b. As in the case of alteration of cell growth and cytotoxicity, the highest proportion of sub-G1 cells was observed when the cells were treated with compound 3b plus R8-PNA-a221. Since the appearance of a sub-G1 population is a hallmark of the activation of apoptosis, an analysis was performed using two apoptosis-associated assays, Annexin V and caspase-3/7 assays.

In order to examine the induction potential of compound 3b and R8-PNA-a221 to induce cell apoptosis and death when used individually and to verify a possible synergistic effect when used in combination, two different apoptosis detection kits were used. Representative plots demonstrating the effects of various concentrations of compound 3b and R8-PNA-a221 administered alone or in combination are shown in Fig. 6. Considering the increase in total apoptotic cells (early and late apoptotic cells of the treated minus the early and late apoptotic cells of the untreated sample or DMSO-treated cells), both agents induced an increase in the number of Annexin V-positive cells in comparison with the controls after 3 days, and this increase occurred in a concentration-dependent manner, reaching a maximum of a 4.82% increase with respect to the control DMSO-treated cells with 6 µM compound 3b and 18.09% with 4 µM R8-PNA-a221 (compared to the untreated cells). Combined treatments with sub-optimal concentrations of compound 3b and R8-PNA-a221 (4 and 2 µM, respectively) led to a sharp induction of apoptosis (13.46%), a proportion which was much higher than the sum of the effects of the singularly added agents (3.06+0.84=4.70%). It should be noted that the increase in the proportion of apoptotic cells was particularly evident in the 'late apoptotic cell' fraction. The increase of the proportion of the 'dead cell' fraction was fully in agreement with the cytotoxicity data depicted in Fig. 4.

The same conclusion derived from the experiments performed using the Annexin V assay can be gathered on the basis of the caspase-3/7 assay shown in Fig. 7. In this case, always considering the increase in total apoptotic cells (early and late apoptotic cells of the treated minus the early and late apoptotic cells of the untreated sample or DMSO-treated cells) obtained following combined treatments with sub-optimal concentrations of compound 3b and R8-PNA-a221 (4 and 2 µM, respectively) was 29.36%, a proportion which was much higher than the sum of the effects of singularly added agents (11.56+4.24%=15.80).

A summary of three independent experiments analyzing the effects of the tetrahydrothieno[2,3-c]pyridine compound 3b and PNA-a221 on cell apoptosis is presented in Fig. 8. In the histogram, only apoptotic cells (early and late) were reported, without including necrotic/dead cells in the calculations; moreover, the percentage of apoptotic cells already present in the untreated samples or in the samples treated with DMSO only was subtracted, in order to better demonstrate the exact increase in apoptotic cells following the treatments. It is evident that the combined treatment led to an increase in apoptosis, which was found to be higher than the sum of the effects obtained using single administration of compound 3b and R8-PNA-a221 (outlined by the dashed lines in the graphs in Fig. 8).

To verify whether the synergistic effect between compound 3b (IC50, 2 µM) and R8-PNA-a221 can also occur in TMZ-resistant T98G cells, the protocol employed on U251 cells and presented in Figs. 6-8 was used. The results shown in Figs. 9 and 10 demonstrated that a synergistic effect between compound 3b and R8-PNA-a221 was also observed on T98G cells. In fact, in both Annexin V (Fig. 9) and caspase-3/7 (Fig. 10) assays, combined treatment led to an increase in apoptosis which was found to be higher than the sum of the effects obtained using single treatment with compound 3b or R8-PNA-a221 (outlined by the dashed lines in Figs. 9B and 10B).