2.2. Results and discussions
2.2.7. Activities of the PITENINs under Cellular Environment
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Figure 2.8. Viability of 2.2 (PIT-1) (A) and 2.5 (DM-PIT-1) (B) measured in HeLa and MDA-MB-231 cells after incubation for 48 h. All MTT assays were performed in triplicates. Viability of HeLa cells was investigated by the MTT assay in the absence and presence of Cl− ions in HBSS buffer in the presence of different concentrations of 2.2 (PIT-1) (C) and 2.5 (DM-PIT-1) (D) after 24 h of treatment. All measurements were performed in triplicates.
16.76 ± 2.47 μM, respectively (Table 2.5). However, compound 2.1 showed poor ion transport efficiency in comparison to 2.2 (PIT-1) and 2.5 (DM-PIT-1), and it also showed very similar cell viabilities as of 2.2 (PIT-1). Hence, compounds 2.5 (DM- PIT-1) and 2.2 (PIT-1) were selected for the further cellular activity studies for their stronger Cl– ion transport efficiency and previously reported apoptosis triggering behavior. A slightly higher cell death rate was observed for 2.2 (PIT-1) than for 2.5 (DM-PIT-1), complying with the higher Cl– ion transportability of 2.2 (PIT-1). The
outcome of the MTT assays is in accordance with the earlier reports. It is well demonstrated that the binding of both 2.5 (DM-PIT-1) and 2.2 (PIT-1) to the PH domain of AKT protein induces apoptosis through the inhibition of the PI3K-PDK- AKT pathway. Cell death in the presence of 2.2 (PIT-1) and 2.5 (DM-PIT-1) showed various apoptotic features. The treatment of 2.2 (PIT-1) resulted in a significant increase in subG1 DNA content both in HeLa and BHK-21 cells. An increase in subG1 and DNA content was also observed in 2.5 (DM-PIT-1) treated BHK-21 cells.
Now to comprehend whether the decrease in cell viability is only due to the suppression of the PI3K-PDK1-Akt pathway or transmembrane Cl– transport by the PITENINs also has a role to play, the viability of the HeLa cells was assessed by changing the extracellular Cl– ion concentrations. Cells were incubated separately in HBSS (Hank’s balanced salt solution), having two different salt compositions (one with a Cl– ion and the other without a Cl– ion). Then, to compare the viabilities of HeLa cells in the different salt compositions of HBSS, cells were incubated with 2.5 (DM- PIT-1) and 2.2 (PIT-1) separately. Lower viability was detected for cells in the Cl– ion-containing buffer as compared to the one in which there was no Cl– ion. This observation validates that the decrease in cell viability is also due to the transport of Cl– ions by 2.5 (DM-PIT-1) and 2.2 (PIT-1) across the cell membrane.
Figure 2.9. Representative images of HeLa cells in the absence and presence of 2.2 (PIT-1) (25 µM) and 2.5 (DM-PIT-1) (50 µM) after 24 h of incubation (A-C). Cells were stained with JC-1 dye. Both red and green channels were merged into each image. Western blot analysis of HeLa cells in the presence of 2.2 (PIT-1) and 2.5 (DM-PIT-1) after 24 h of treatment (D). The PARP and β-actin antibodies were used to develop the Western blot.
The difference between the cell viability in HBSS buffer in the presence and absence of a Cl– ion is in accordance with earlier reports. However, it is incredibly challenging to prepare a complete Cl– ion free extracellular environment because
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intracellular Cl– ions can also efflux out to balance the Cl– ion gradient across the membrane. The excessive imbalance of the Cl– ion gradient can lead to cell death. The interference of the mitochondrial membrane potential (MMP) also suggests the Cl– ion transport efficacies of the compounds (Fig. 2.9). We also performed the HPTS quenching assay using giant unilamellar vesicles (GUV). The result of the HPTS quenching assay disclosed that the-presence of the compound significantly induces the influx of Cl– ions (Fig. 2.10, S2.16).confirmed that the transport of Cl– ions results in faster quenching of the encapsulated HPTS fluorescence. Therefore, the MTT assay in HBSS media and GUV-based HPTS quenching assays propose that the 2.5 (DM- PIT-1) mediated transport of the Cl– ion enhanced the intracellular Cl– ion concentration, and this higher intracellular Cl– ion concentration is directly related to the viability of the HeLa cells. Other research groups and we already showed that chloride transport causes an imbalance in ionic homeostasis in cells, which in turn induces apoptotic cell death. It is well documented
Figure 2.10. Green channel and bright‐field images microscopic images of the HPTS‐
encapsulated GUVs before (A, C) and after 2 min of the addition of 0.1 mM of 2.5 (DM- PIT-1) and NaCl (B, D).
that the transport of Cl– ion disrupts the mitochondrial membrane potential and persuades the cytochrome c release from mitochondria. The released cytochrome c forms a cytochrome c/Apaf-1 complex, which stimulates numerous caspase-9- mediated pathways to persuade cellular apoptosis. Hence, the apoptotic process can be scrutinized by investigating the change in mitochondrial membrane potential and concurrent release efficiency of cytochrome c, level of expression of caspases, and extent of nuclear fragmentation. The family of poly(ADP-ribose) polymerase (PARP) proteins is associated with several cellular functions including expression of crucial genes, DNA repair, apoptosis, and others. Meanwhile, the existence of cleaved PARP protein is regarded as a trademark of apoptosis in the presence of active caspases.
The presence of cleaved PARP (Fig. 2.10) confirmed the caspase-mediated apoptotic cell death in the presence of 2.2 (PIT-1) and 2.5 (DM-PIT-1). Inhibition of the PIP3/AKT interactions also promotes cancer cell death through an apoptotic pathway. However, it is difficult to estimate/quantitatively measure the extent of cell death caused by the inhibition of the PIP3/AKT interaction or by transmembrane transport of Cl– ions by the compounds, which is beyond the scope of this investigation. Overall, the results suggest the transmembrane Cl– ion transportability of the PITENINs.
Overall, the current study describes a different approach to PITENINs-mediated regulation of cellular AKT signaling, targeting transmembrane Cl– ion transport. Due to the different mode of actions, the PITENINs can be combined with the existing selective AKT kinase inhibitor to accomplish greater efficacy in promoting apoptosis in cancer cells. The different modes of action of PITENINs may assist in blocking PIP3
signaling in combination with the PI3K inhibitors because of the inactivation of PIP3
phosphatase in the breast, endometrium, colon, and other cancer cells. These dual activities of PITENINs may also represent a promising strategy to fight against PIP3
phosphatase-deficient tumors. However, further studies are required to understand the role of PITENINs in other cancer cells. Target specific delivery of PITENINs to these PIP3 phosphatase-deficient cancer cells could also limit side effects related to
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the inhibition of the PI3K signaling pathway. Furthermore, PITENINs with such different modes of action could be an attractive class of lead compounds for anticancer drug discovery.