MBZ and ABZ lower cancer cell viability, especially in colorectal cancer cell line HT29
The IC50 values of MBZ and ABZ over 48 hours were determined in three cancer cell lines MCF-7, MDA-MB-231 (both breast cancer) and HT-29 (colon cancer), as well as in the non-transformed mesenchymal stromal-cell line RCB2157, using MTT cell viability assays (Fig 1a-d). The IC50 values in the mesenchymal stromal cell line RCB2157 were the highest by several fold (Table 1). The two breast cancer lines were less sensitive to the two drugs than the colorectal cancer cell line HT29.
MBZ and ABZ block the cell cycle at G2/M
Further analyses were carried out in the HT29 cells to determine the mechanism of the loss of cell viability after 24 or 48 hours treatment with MBZ or ABZ. Progression through the cell cycle was analysed using fixation and staining with propidium iodide (PI). Paclitaxel, known to cause a block in the cell cycle at G2/M through binding to microtubules, was used as a positive control.
Treatment with 2 µM MBZ or ABZ or 50 nM paclitaxel resulted in significantly large increases in the G2/M phase compared to control (Fig. 1e-f). At 24 hours, MBZ was at 60.18%, ABZ at 58.68% and paclitaxel at 62.42% compared to vehicle-only control at 21.37% (P > 0.05). There were smaller, but still significant increases of cells in the >G2/M phase, and concomitant decreases in the proportion of cells in the G1 and S phases.
MBZ and ABZ induce apoptotic nuclear morphology and depolymerise microtubules
In order to investigate whether MBZ and ABZ interact with microtubules in a similar way to paclitaxel, HT-29 cells were stained for nucleus and microtubules following treatment with MBZ (2 µM), ABZ (2 µM), paclitaxel (50 nM) or DMSO vehicle control (0.1% v/v) for 8 hrs. Hoechst 33342 staining was used for the nucleus, and antibodies against either -tubulin, a main component of the tubulin dimer that polymerises to form microtubules or against acetyl-α-tubulin, an indicator of stabilised microtubules, were used to visualise microtubule structure .
MBZ, ABZ and paclitaxel treatments all produced the condensed nuclei characteristic of apoptosis. α-tubulin and acetylated-α-tubulin both displayed a decreased fluorescent intensity following MBZ and ABZ treatment compared to control. By contrast, paclitaxel increased intensity of both α- and acetylated-α -tubulin staining compared to control. This suggests that MBZ and ABZ promote depolymerisation of microtubules, whereas paclitaxe l promotes polymerisation and stabilises microtubules, as expected.
MBZ and ABZ induce caspase-3 activity
Since it appeared that cell death was occurring concurrently with a block in the cell cycle, characteristics associated with various forms of apoptosis were investigated. Caspase-3 activity was measured in HT-29 cells following treatment with MBZ, ABZ, paclitaxel or vehicle-only control as before (Fig. 2a). Both untreated control and DMSO vehicle control showed low levels of activity. ABZ and MBZ treatment both increased caspase activation around 550-fold at 24 hours (P < 0.0001 compared to untreated control), which dropped back to 300-400 fold at 48 hours, suggesting that the cell death process had peaked within the first 24 hours. Paclitaxel treatment caused the greatest increase caspase activity, up to 750-fold of control at 24 hours (P < 0.0001 compared to MBZ, ABZ and controls), which is not surprising as paclitaxel is a classical apoptosis inducer and activates caspase-3 .
MBZ and ABZ cause early phosphatidylserine exposure
Following both 24 and 48 hr treatments, cells were double-stained with fluorescently labelled Annexin-V to identify early externalisation of phosphatidylserine residues, and PI to separate out the internal phosphatidylserine exposed to Annexin-V by cell membrane permeability, occurring as a result of primary or secondary necrosis (Fig. 2b,c). Following 24hr treatment, MBZ and ABZ caused approximate 25% decreases in the percentage of viable cells (negative for both Annexin V and PI) compared to control (P < 0.0001), similar to that of the positive control paclitaxel (Fig. 2b). MBZ and ABZ caused a significant increase in the percentage of cells with phosphatidylserine exposure, with 22.83% of MBZ treated cells and 24.17% of ABZ treated cells positive for Annexin V only, similar to paclitaxel at 20.72%, whereas only 3.37% of control cells were positive (P < 0.0001). Numbers of double stained cells undergoing secondary necrosis after treatment with paclitaxel, MBZ and ABZ all significantly increased to MBZ 5.78%, ABZ 5.99% and paclitaxel 7.94% vs control 2.12% (P < 0.0001). Necrotic cells, positive for PI only, were increased only in MBZ and ABZ treatments compared to control, 3.58% and 3.48% vs 0.54% respectively. Forty-eight-hour treatments produced similar results to that of 24hr treatments.
MBZ and ABZ treatment results in DNA fragmentation
Another indicator of apoptosis is the fragmentation of DNA content, known as sub-G1 DNA, measured using fixation of cells and PI staining. Here, following both 24 and 48hr treatments with MBZ, ABZ and paclitaxel, the percentage of cells with sub-G1 DNA content was significantly increased compared to control (Fig. 2d). After 24hrs, MBZ treatment resulted in 13.84% (P = 0.001) of cells with sub-G1 DNA content, ABZ 11.53% (P = 0.026), paclitaxel 19.33% (P < 0.0001) and control 6.45%. DMSO caused no statistically significant change. Following 48hr treatment, all amounts increased, except in the controls.
MBZ and ABZ increase mitochondrial membrane permeability
HT-29 cells were treated with MBZ, ABZ, paclitaxel, DMSO or left untreated for 24 and 48 hrs and the percentage of cells with higher mitochondrial membrane permeability, indicative of apoptosis, determined using the mitochondrial-targeted stain TMRE.
In the 24hr treatments, there were no significant differences between DMSO vehicle control (3.49 ± 0.38%) and control (3.36 ± 0.29%, P > 0.05). However, in MBZ, ABZ and paclitaxel treatments there were approximate 6-fold increases in the percentage of cells with permeabilised mitochondrial membranes (Fig. 2e). Cells exposed to the treatments for 48 hrs exhibited similar results, suggesting that the majority of cells had passed through the earlier phases of apoptosis by this time.
MBZ and ABZ induce oxidative stress, resulting reduced thiol oxidation
Indirect measurement of reactive oxygen species damage was undertaken using the DTNB assay, which quantifies reduced thiol content, from both protein and glutathione sources. This decreases as a consequence of oxidative stress. Following 48hr treatments, the reduced thiol concentration was significantly decreased in cells treated with MBZ, to 2.671 x 10-5 M (P = 0.0094), ABZ to 2.646 x 10-5 M (P = 0.0086) and paclitaxel to 2.539 x 10-5 M (P = 0.0058) in comparison to the control at 4.630 x 10-5 M (Fig. 2f).