Pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs) exert their anti-proliferative activity by interfering with Akt-mTOR signaling and bax:bcl-2 ratio modulation in cells from chronic myeloid leukemic patients

Abstract

In our study we found that pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs) mediated apoptosis in primary leukemia cells from 27 chronic myelogenous leukemia (CML) patients at onset through the activation of the caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP). The bax:bcl-2 ratio was increased as a consequence of down-regulation of bcl-2 and up-regulation of bax proteins in response to treatment with PBTDs. In addition, PBTDs were able to induce cell death in primary leukemia cells derived from 23 CML-chemoresistant patients. Furthermore, the effects of PBTDs on the Akt-mTOR (mammalian target of rapamycin) pathway were determined by Western blot. PBTDs possessed inhibitory activity against mTOR and also impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. The results presented in this study demonstrate that we have identified the PBTDs as restoring the apoptotic pathways both in primary leukemia cells derived from CML patients at onset and in primary leukemia cells derived from CML-chemoresistant patients, thus showing their ability to undergo apoptosis. These compounds constitute a promising therapeutic approach for patients with leukemia. They provide the basis for new strategies for an additional anticancer drug in leukemia therapies, especially when conventional ones fail.

Figure 1

The chemical structure of the compounds.

Figure 2

Pyrrolo[1,2‐b][1,2,5]benzothiadiazepines (PBTDs) effects in primary leukemia cells isolated from chronic myelogenous leukemia (CML) patients examined for DNA fragmentation. DNAs were extracted from untreated primary leukemic cells (lanes cc) and treated primary leukemic cells. Agarose gels are representative of at least three separate experiments.

Figure 3

Pyrrolo[1,2‐b][1,2,5]benzothiadiazepines (PBTDs) effects in primary leukemia cells isolated from chronic myelogenous leukemia (CML) patients examined for caspase cascade activation. Whole‐cell lysates were prepared from untreated primary leukemic cells (lanes cc) and treated primary leukemic cells. Cellular extracts were analyzed by Western blot using anti‐caspase‐8, ‐3 antibodies. β‐Actin was used as a control for protein loading. The molecular weight (in KDa) of protein size standards is shown on the left‐hand side. Blots are representative at least three separate experiments.

Figure 4

Pyrrolo[1,2‐b][1,2,5]benzothiadiazepines (PBTDs) effects in primary leukemia cells isolated from chronic myelogenous leukemia (CML) patients examined for caspase cascade activation. Whole‐cell lysates were prepared from untreated primary leukemic cells (lanes cc) and treated primary leukemic cells. Cellular extracts were analyzed by Western blot using anti‐caspase‐9, ‐3 antibodies. β‐Actin was used as a control for protein loading. The molecular weight (in kDa) of protein size standards is shown on the left‐hand side. Blots are representative at least three separate experiments.

Figure 5

Pyrrolo[1,2‐b][1,2,5]benzothiadiazepines (PBTDs) effects in primary leukemia cells isolated from chronic myelogenous leukemia (CML) patients examined for bcl‐2 protein family. (A) Whole‐cell lysates were prepared from all untreated primary leukemic cells (lanes cc) and treated primary leukemic cells at indicated times. Western blot analysis was performed with antibodies which specifically recognized bax, bcl‐2, and β‐actin. β‐Actin was used as an internal loading control. The molecular weight (in KDa) of protein size standards is shown on the left‐hand side. Blots are representative of at least three separate experiments. (B) PBTDs effects in primary leukemia cells isolated from CML patients examined for bax:bcl‐2 ratio. Bax:bcl‐2 ratio in untreated primary leukemic cells (lane cc) and treated primary leukemic cells was obtained by densitometry analysis of the blots shown in Figure 6. Significant differences between the primary leukemic cells treated with PBTDs and untreated primary leukemic cells are indicated by probability P. *P < 0.05, **P < 0.01, and ***P < 0.001; Student’s t‐test.

Figure 6

Translocation of bax from the cytosol to the mitochondria in patient UPN37 treated with RS 678, RS 779, and RS 2630. Cell lysates were collected and equal amounts of protein from each sample were subjected to Western blot analysis and probed for bax. β‐Actin was used as an internal loading control. The molecular weight (KDa) of protein size standards is shown on the left‐hand side.

Figure 7

Pyrrolo[1,2‐b][1,2,5]benzothiadiazepines (PBTDs) effects in primary leukemia cells isolated from chronic myelogenous leukemia (CML) patients examined for poly(ADP‐ribose) polymerase (PARP) cleavage. Whole‐cell lysates were prepared from untreated primary leukemic cells (lanes cc) and treated primary leukemic cells. Cellular extracts were analyzed by Western blot using specific antibody. β‐Actin was used as a control for protein loading. The molecular weight (in KDa) of protein size standards is shown on the right‐hand side. Blots are representative at least three separate experiments.

Figure 8

Pyrrolo[1,2‐b][1,2,5]benzothiadiazepines (PBTDs) effects in primary leukemia cells isolated from chronic myelogenous leukemia (CML) patients examined for Akt–mTOR signaling. Whole‐cell lysates were prepared from all untreated primary leukemic cells (lanes cc) and treated primary leukemic cells at indicated times. Western blot analysis was performed with specific antibodies. Blots are representative at least three separate experiments.