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.
The American mayapple (Podophyllum) has been a source of bio-active lignans for many years. The most studied one of these is podophyllotoxin (1)
This natural compound 1 presents a chemical structure consisting of four fused planar rings with consecutive chiral centers [
Arguably the most important derivative of these has been etoposide (also known as VP-16). This drug has been widely studied and has been classified since the 1980’s as a topoisomerase II inhibitor [
The success of etoposide has led to a great deal of work with podophyllotoxin derivatives, much of it studying a variety of biological effects and anti-cancer activities [
Synthesis of podophyllotoxin in laboratory is still quite challenging chemistry [
In our previous work we explored the synthesis and application of novel aza-podophyllotoxin derivative compounds which led to several new analogs with anti-cancer potential [
Expression of phosphatidylserine was measured as an apoptosis marker. Both tested compounds presented statistical significance (P < 0.05) for apoptosis induction. Compound 8a presented 44% of apoptotic cells while 9a presented 59.5%.
apoptotic stage (50%) shown on
Cells with fragmented DNA were assessed using DAPI staining. Results indicate significant fragmentation for both tested compounds.
We analyzed the cell cycle to assess possible details which may give insight to the mechanism of action of the tested compounds. Our results indicate that the majority of cells exposed to the tested compounds were arrested at the S phase of the cell cycle (
Mitochondrial mediated apoptosis can be an indicator of intrinsic apoptotic pathway. We examined this phenomenon to assess the possibility of an intrinsic mechanism after exposure to our tested compounds. Our results indicate that only compound 9a induced a statistically significant (P < 0.05) permeabilization of the mitochondrial membrane (MM). Compound 9a caused a mean of 82.5% (
Caspase activation can provide insight in the mechanism of action and cell death processes. We analyzed caspase 3 and 7 effector caspases after exposure to our tested compounds. Results show (
Caspase 8 was measured to determine if apoptosis occurs via an intrinsic or extrinsic pathway. Our results show that only compound 9a caused significant activation of caspase 8 with an 82.5% of cells with activated caspase 8 (
Caspase 9 activation is a hallmark event of the intrinsic apoptotic pathway. Our results show activation of caspase 9 on cells treated with the 9a compound (89.5%). This was unexpected since caspase 8 was also activated thus an extrinsic mechanism was presumed to be involved (
In this study we screened the biological activity and apoptosis induction of two novel aza-podophyllotoxin derivatives. These compounds were previously assayed for toxicity against a number of cell lines and were determined to be active for growth inhibition upon COLO 205 colorectal adenocarcinoma cells among other cancer cell types. Both of our tested compounds presented different biological activities on the COLO 205 cell line. Com-
pound 8a presented an unexpected apoptotic activity profile. This compound presented PS migration to the exterior of cells as evidenced by the annexin V staining. Migration of PS is usually indicative of an apoptotic cell death mechanism. Additionally this compound presented a significant cell cycle arrest at the S phase and as well as DNA fragmentation; however none of the traditional apoptosis hallmarks such as mitochondrial membrane permeabilization and caspase 8, 9, 3 and 7 activation were detected in a significant manner.
This type of apoptosis sometimes referred to as atypical apoptosis, has been observed after activation of intra-S DNA damage checkpoints [
Our results for compound 8a are similar to previous work [
Although a complete mechanistic description was beyond the scope of this work, we found interesting evidence which suggests a structure-activity difference for our aza-podophyllotoxin derivatives. Compound 8a presents similar core structure to the podophyllotoxin, precursor of etoposide. The only difference is substitution of nitrogen in ring “C” at position 4, elimination of two chiral centers and elimination of 2 oxygen atoms from ring A. Substitution of nitrogen in ring C might not make significant difference in biological activity [
This project was supported by the National Center for Research Resources and the National Institute of General Medical Sciences of the National Institutes of Health through Grant Number 8 P20 GM 103475.