Biological Activity of N-Hydroxyethyl-4-aza-2,3-didehydropodophyllotoxin Derivatives upon Colorectal Adenocarcinoma Cells

Abstract

Etoposide is a chemotherapy drug derived from the natural lignin podophyllotoxin. Our novel generated Aza-podophyllotoxin compounds (AZP 8a & AZP 9a) are analogues of podophyllotoxin and were previously screened for anti-cancer activity through the NCI 60 cell line screening panel showing activity on various cell types including colon cancer. This study expands the toxicological screening by studying apoptosis and various hallmark events as part of the mechanism of action of these compounds on colon cancer cells. The COLO 205 cell line was selected and exposed to AZP to determine the IC50 doses at 24 hours treatment. Apoptosis hallmark events such as migration of phosphatidylserine (PS) to the cell membrane, DNA fragmentation, cell cycle effects, mitochondrial membrane permeabilization and caspase activation were included. Experiments were performed in triplicates for all tested compounds including AZP 8a, AZP 9a, camptothecin as positive control and vehicle as negative control. Our results present contrasting apoptotic activity between the experimental compounds. Compound 8a presented migration of PS (annexin V assay), DNA fragmentation and cell cycle arrest at S phase. Compound 9a presented PS migration with fragmented DNA, cell cycle arrest at S phase, mitochondrial membrane permeabilization and activation of caspase 3, 8 and 9. Compound 8a without the oxygen atoms in ring A appears to cause effects similarly to autophagy as induced by etoposide, a cancer drug analogue of our heterocyclic compounds. Compound 9a with the oxygen atoms in expanded ring A presented induction of cell death following activation of a classical apoptosis pathway. Our results suggest that minor structural differences among these AZP can account for the difference in biological response and cancer cell toxicity.

Keywords: Aza-Podophyllotoxin; COLO 205; Colon Cancer; Etoposide; Podophyllotoxin.

Figure 1

(a) Structure of Podophyllotoxin 1; (b) Drugs Derived from Podophy llotoxin; (c) N-Hydroxyethyl-4-aza-didehyropodophyllotoxin Derivatives.

Figure 2

(a) Annexin V analysis. Results clearly indicate apoptotic activity for both compounds. 8a presents 44% of apoptotic cells whereas 9a presents 59.5%. Both compounds present higher activity than the positive controls etoposide (32%) and podophyllotoxin (27%); (b) Annexin V Staining Histogram. Data presents the stage of apoptosis after exposure to the tested compounds. Compound 9a presents the majority of cells (50%) at a late apoptotic stage (top right quadrant). Positive controls and test compound 8a present mostly cells at an early apoptotic stage clustered in the bottom right quadrant.

Figure 3

DNA Fragmentation. Results indicate significant DNA fragmentation for both tested compounds 8a (20.9%) and 9a (30.5%) in comparison with the analog controls etoposide (9.5%) and podophyllotoxin (16%).

Figure 4

Cell Cycle. Results indicate that the majority of cells exposed to the tested compounds were arrested at the S phase. The majority of the cells exposed to compound 8a were arrested at the S phase followed by 13% at the G2/M, 10% at the Sub G0 and 3% at the G0/G1 stage. For compound 9a the majority of cells (52%) were arrested at the S phase followed by 12% at the sub G0 stage, 8% at the G2/M and 2% at the G0/G1 stage.

Figure 5

Mitochondrial Membrane Permeabilization. Results indicate that compound 9a caused significant mitochondrial permeabilization, but not 8a. Compound 9a caused a mean of 82.5%, compound 8a 41%, the controls etoposide 35.5%, podophyllotoxin 37.5% and the vehicle 21% mitochondrial membrane permeabilization.

Figure 6

(a) Caspase 3 and 7 activation. Results reveal that among the experimental drugs compound 9a caused significant activation of effector caspases in contrast to the compound 8a; (b) Caspase 3 and 7 activation. Compound 9a caused significant activation of effector caspases in contrast to compound 8a. As observed the majority of cells are in the late apoptotic stage (upper right quadrant). Caspase activated green fluorescent cells (lower right box) were used in this assay.

Figure 7

Caspase 8 activation. Results show that compound 9a activated caspase 8 on treated cells in contrast to the compound 8a where no significant activity was detected.

Figure 8

Caspase 9 activation. when comparing our test drugs, only compound 9a induced significant activation of caspase 9 in contrast to compound 8a.