Adamantyl-substituted retinoid-related molecules induce apoptosis in human acute myelogenous leukemia cells

Abstract

The adamantyl-substituted retinoid-related (ARR) compounds 3-Cl-AHPC and AHP3 induce apoptosis in vitro and in vivo in a newly established human acute myelogenous leukemia (AML) cell line, FFMA-AML, and in the established TF(v-SRC) AML cell line. FFMA-AML and TF(v-SRC) cells displayed resistance to apoptosis mediated by the standard retinoids (including trans-retinoic acid, 9-cis-retinoic acid, and the synthetic retinoid TTNPB) but showed sensitivity to apoptosis mediated by 3-Cl-AHPC- and AHP3 in vitro and in vivo as documented by poly(ADP-ribose) polymerase (PARP) cleavage and apoptosis terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay. 3-Cl-AHPC or AHP3 exposure in vitro resulted in decreased expression of the antiapoptotic proteins (cellular inhibitor of apoptosis 1, X-linked inhibitor of apoptosis protein) and phospho-Bad and activated the NF-κB canonical pathway. A significant prolongation of survival was observed both in nonobese diabetic severe combined immunodeficient mice carrying FFMA-AML cells and treated with either 3-Cl-AHPC or AHP3 and in severe combined immunodeficient mice carrying TF(v-SRC) cells and treated with AHP3. We have previously shown that ARRs bind to the orphan nuclear receptor small heterodimer partner (SHP) and that the expression of SHP is required for ARR-mediated apoptosis. Induced loss of SHP in these AML cells blocked 3-Cl-AHPC- and AHP3-mediated induction of apoptosis. These results support the further development of 3-Cl-AHPC and AHP3 as potential therapeutic agents in the treatment of AML patients.

Figure 1

Apoptosis induction in FFMA-AML and TF(v-SRC) cells by 3-Cl-AHPC and AHP3. (A) Structures of 3-Cl-AHPC and AHP3. (B) Cells were seeded at 1 × 10⁴ cells/ml and grown for 24 h, then exposed to varying concentrations of AHP3 or 3-Cl-AHPC for 96 h, (C) incubated with AHP3 or 3-Cl-AHPC (1 μM) for 0 to 96 hours and (D) exposed to 1 μM all trans retinoic acid, 9-cis-retinoic acid and TTNPB. The percentage of apoptotic cells was determined using acridine orange and ethidium bromide staining as described in Matrials and Methods. The error bars represent the mean of three separate determinations +/− the standard deviation (SD).

Figure 2

Induction of apoptosis and 3-Cl-AHPC and AHP3 mediated caspase-3 activation and cleavage of caspase-3. FFMA-AML and TF(v-SRC) cells were exposed to 1 μM 3-Cl-AHPC and AHP3. (A) Apoptosis in FFMA-AML and TF(v-SRC) cells following 24 and 48 h ARR exposure, respectively. (B) Percentage of apoptotic cells for indicated times. Induction of apoptosis and cell death was assessed using Annexin V-FITC labeling with propidium iodide (PI) staining; the percentage of apoptotic cells corresponds to the sum of percent noted in upper right (late apoptotic cells, annexin V and PI positive cells) and lower right (early apoptotic cells, annexin V positive, PI negative) quadrants. (C) Activation of caspase-3 in FFMA-AML and TF(v-SRC) cells following exposure to ARR for varying times. (D) Generation of caspase -3 (17 kDa) fragment and caspase-3 protein levels. Caspase-3 activation was determined as described in Methods. The error bars represent the mean of three separate determinations +/− the standard deviation. *, ** and *** were significantly different in comparison to control cells. (p value is <0.05, <0.01 and <0.001 as determined by t test).

Figure 3

AHP3-mediated inhibition XIAP, c-IAP1, and phospho-Bad expression in FFMA-AML and TF(v-SRC)) cells accompanied by the induction of PARP cleavage, AHP3 and 3-Cl-AHPC induce NF-κB activation in canonical pathway. (A) AHP3 decreased the expression of XIAP, c-IAP1 and phospho-Bad protein in Western blot. (B) AHP3 and 3-Cl-AHPC enhanced the IκBα degradation, and (C) NF-κBp65(Ser276) phosphorylation in AML cells. (D) 3-Cl-AHPC enhanced IKKα (Ser180) and IKKβ (Ser181) phosphorylation/activation in cells (left panel). NF-κB inhibitor, JSH-23 inhibited ARR mediated apoptosis in TF(v-SRC) cells (right panel); cells were treated for 48 h. * and ** significantly different in comparison to control cells (p value is <0.05 and <.01 as determined by t test). The error bars represent the mean of three separate determinations +/− the standard deviation.

Figure 4

Loss of SHP expression inhibited 3-Cl-AHPC-induced apoptosis and in AML cells. (A and B) Knockdown of SHP expression inhibited 3-Cl-AHPC mediated apoptosis in FFMA-AML and TF(v-SRC) cells. FFMA-AML and TF(v-SRC) cells were transfected with shRNA SHP retroviral expression vectors for 48 h, then cells were exposed to 1 μM 3-Cl-AHPC for 15 and 24 h, respectively. Apoptosis was assessed by flow cytometry using Annexin V-FITC and propidium iodide. (C) SHP knockdown blocked the 3-Cl-AHPC and AHP3 mediated inhibition of proliferation in TF(vSRC) cells. The error bars represent the mean of three separate determinations +/− the standard deviation. * significantly different in comparison to sh-vector treated cells (p value is <0.05 as determined by t test).

Figure 5

AHP3 and 3-Cl-AHPC inhibition of FFMA-AML proliferation in NOD-SCID mice, prolongation of survival and AHP3 inhibited TF(v-SRC) proliferation and growth in SCID mice. (A) Sixteen NOD-SCID mice were randomly divided into two groups of eight mice each and were injected through the tail vein with 1 ×10⁶ FFMA-AML cells. Treatment with either vehicle or 3-Cl-AHPC (30 mg/kg) given twice intravenously daily for 4 days was instituted 24 h following injection of the cells (left panel). An second study, identical to the one described above, was performed with 3-Cl-AHPC by an interperitoneal route (A, right panel). (B) Twenty four NOD-SCID mice were randomly divided into three groups of eight mice each. Each mouse was injected with 1 × 10⁶ FFMA-AML cells; 24 h later after injection, treatment was started with either vehicle or AHP3 given at doses 7.5 mg/kg or 5.0 mg/kg intravenously twice daily through the tail veins for 5 days. Mice were then observed daily for evidence of toxicity and prolongation of survival time. (C) Survival of TF(v-SRC) inoculated mice treated with AHP3. NOD SCID mice were randomly divided into two groups of eight mice. Each mouse was injected with 1 × 10⁷ TF(v-SRC) cells and 24 h later mice were either treated intravenously with AHP3, 20 mg/kg or vehicle on a Monday, Wednesday and Friday schedule for a total of 15 injections. Mice were observed daily for evidence of toxicity and prolongation of survival. Absence of weight loss in AHP3 treated SCID mice (C, right panel). (D) AHP3 inhibited palpable TF(v-SRC) growth in SCID mice. TF(v-SRC) cells were grown and maintained subcutaneously in a SCID mouse. Tumors were harvested and approximately 20 mg of tumor was subcutaneously trochared into each flank of a SCID mouse. The tumors were then allowed to reach a size of 100 mg and the mice randomly divided into three groups of 8. Groups were administered 20 mg/kg AHP3 either intravenously (I.V.) or subcutaneously (S.C.) (at a site distant from the tumor) on a Monday, Wednesday and Friday scheduled for 12 injections. Control mice were treated intravenously with vehicle. The error bars represent the standard deviation.

Figure 6

AHP3 mediated apoptosis and PARP cleavage in treated tumor tissues. (A) Size of untreated and AHP3 treated paraffin embedded tumor tissue sections in millimeter. (B) DNA strand breaks in tumors obtained from vehicle and AHP3 intravenously (I.V.) or subcutaneously (S.C.) treated mice were detected by TUNEL assay using the in situ detection kit. (C) TUNEL positive cells in tumor tissues. (D) Immunohistochemical staining of paraffin-embedded tissue sections for cleaved PARP. Apoptotic cells were indicated by arrow. Details of slides preparation, visualization and antibody utilized are described in Materials and Methods.