3D inhibits cell viability
Treatment of HT-29 cells with 3D (Figure 1A) for 24 h resulted in the inhibition of cell viability in a dose-dependent manner. The IC50 was found to be 10 μM in HT-29 cells (Figure 1B). Similar results were obtained in SW620, another human CRC cell-line with the IC50 calculated to be 6.25 μM (Figure 1C). These findings were further confirmed using the xCELLigence real-time cell proliferation system. 3D was found to inhibit the cell proliferation in a dose- and time-dependent manner (Figure 1D). These findings indicate that 3D significantly inhibited viability of CRC cells.
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3D induces ROS-mediated apoptosis
Most of the inhibition in cell viability occurs due to apoptosis or necrosis. To understand whether 3D induces apoptosis or necrosis, SW620 cells treated with different concentrations of 3D resulted in a significant induction of apoptosis and lesser necrosis (Figure 2A). Chronic accumulation of intracellular ROS induces cell death. Preincubation with an antioxidant, N-acetylcysteine (NAC), decreased apoptosis indicating that 3D-induced apoptosis is ROS dependent (Figure 2B). A similar result was obtained in HT-29 cells (Figure 2C). We examined the effect of 3D on ROS production. Treatment of HT-29 with 3D resulted in enhanced production of ROS in a dose-dependent manner (Figure 2D). Similarly, 3D also induced ROS production in SW620 cells (Figure 2E). Most of the intracellular ROS generation takes place from mitochondria. Treatment of human CRC cell lines with 3D resulted in the decrease of mitochondrial membrane potential (Figure 2F, left). A similar result was obtained in SW620 cells (Figure 2F, right). These results demonstrated that 3D induces ROS production, inhibits mitochondrial membrane potential and enhances ROS-dependent apoptosis.
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3D induces p53, modulates Bcl2 family proteins and induces cytochrome and PARP activation
p53 is a tumor suppressor gene and acts through several mechanisms for anticancer effect. It activates DNA repair proteins and induces growth arrest, apoptosis and senescence. 3D treatment resulted in the induction of p53 expression in HT-29 cells (Figure 3A). Bax is a p53 target gene, and as expected, treatment of HT-29 cells with different concentrations of 3D induced Bax expression (Figure 3A). Similar results were obtained in SW620 cells (Figure 3B). The balance between Bax and Bcl2/BclxL determines the cell fate under stress. Treatment with 3D resulted in the inhibition of Bcl2 and BclxL in HT-29 (Figure 3A) and SW620 (Figure 3B) cells. Thus, 3D was found to inhibit antiapoptotic proteins and induce proapoptotic proteins, thereby triggering the apoptotic pathway. Upon initiation of apoptosis signaling, activated Bax binds to the mitochondrial outer membrane and is known to induce the opening of the mitochondrial voltage-dependent anion channel (VDAC) that would lead to the release of cytochrome c from mitochondria into cytosol where it activates the caspase cascade. Indeed, treatment with 3D resulted in the release of cytochrome c in HT-29 cells (Figure 3C). Similar results were obtained in SW620 cell line (Figure 3D). PARP family of proteins is known to be involved in DNA repair and programmed cell death. Our results further demonstrated that treatment with 3D resulted in the cleavage of PARP as shown by an increase in cleaved PARP in HT-29 cells (Figure 3C). Similar results were obtained in SW620 cells (Figure 3D).
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3D activates caspase cascade
Release of cytochrome c from mitochondria into cytosol leads to caspase activation that plays an essential role in apoptosis. To determine which caspases were involved in inducing apoptosis, 3D-treated HT-29 cells were incubated with various caspase substrates. 3D was found to activate caspase-9, caspase-6 and caspase-3 and to lesser extent caspase-8 as well (Figure 4A). Similar finding was obtained in SW620 cells (Figure 4B). Thus, these results indicate that 3D activated mostly those caspases that are involved in intrinsic apoptotic pathway. However, some induction of caspase-8 was also observed, which is important for extrinsic apoptotic pathway.
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