AKT Pathway is Activated by 1,25-Dihydroxyvitamin D3 and Participates in its Anti-Apoptotic Effect and Cell Cycle Control in Differentiating HL60 Cells

Abstract

Differentiation therapy for cancer is a developing treatment modality that is based on the anti-proliferative effects associated with differentiation of the malignant cells. 1,25-dihydroxyvitamin D 3 (1,25D) and its analogs are currently being evaluated clinically, alone or in combination with other agents, for treatment of several neoplastic diseases, but their usefulness as single agents may be limited by the enhancement of cell survival in some cell types exposed to 1,25D. In this study we evaluated the role of AKT signaling pathway, known to be anti-apoptotic in diverse cell types, in enhancing the survival of human leukemia HL60 cells induced to differentiate with 1,25D. We found that the phosphorylation and activity of AKT, as well as of its downstream targets, are increased after the exposure to 1,25D. Treatment of HL60 cells with PI3K inhibitors LY294002 and Wortmannin, which decrease the activity of the AKT pathway, induced apoptosis, but this effect was reduced in cells simultaneously treated with 1,25D. Interestingly, LY294002 and Wortmannin also accentuated the 1,25D-induced G 1 to S phase cell cycle block in HL60 cells, and this was associated with an increased expression of p27Kip1. Thus, a combination of 1,25D with inhibitors of AKT pathway is strongly anti-proliferative and should therefore be considered for differentiation therapy of myeloid leukemia.

Raf‐1 signaling is required for the later stages of 1,25‐dihydroxyvitamin D3‐induced differentiation of HL60 cells but is not mediated by the MEK/ERK module

“…These include the JNK (Wang and Studzinski, 2001b;Ji et al, 2002;Wang et al, 2003) and the AKT (Zhang et al, 2006) pathways, and as described here, the Raf-1/p90RSK pathway. It is important to elucidate the precise detailed steps of this pathway in leukemia cells, as it has potential to be targeted for therapeutic intervention (Milella et al, 2001;Chang et al, 2003;Konopleva et al, 2005), and it is essential to identify the correct targets.…”

“…For instance, we and others have shown that in HL60 cells ERK1/2 is activated in the early phase of 1,25D-induced differentiation, and this can be accentuated by serum starving the cells prior to exposure to 1,25D (Marcinkowska et al, 1997;Marcinkowska, 2001;Wang and Studzinski, 2001a). Other studies have implicated JNK (Ji et al, 2002;Wang et al, 2003;Buitrago et al, 2006), p38 (Wang and Studzinski, 2001a,b;Ji et al, 2002) and AKT (Zhang et al, 2006) pathways in the transmission of the 1,25D-generated signals to the transcriptional machinery in the nucleus. However, the Raf-MEK-ERK pathways remains firmly established as a principal all-purpose signaling cascade (Chang et al, 2003), and as such deserve additional attention regarding its role in the induction of differentiation.…”

“…These effects occurred in the time frame of seconds and minutes, but have not been universally observed in other cell types. More recently, studies in leukemia cells showed involvement of several more slowly activated MAPK cascades in signaling of 1,25D effects on cell proliferation (Wang and Studzinski, 2001a; Ji et al., 2002), survival (Wang andPepper et al, 2003;Zhang et al, 2006), and differentiation (Wang et al, 2000;Marcinkowska, 2001;Wang and Studzinski, 2001a;Studzinski et al, 2005). For instance, we and others have shown that in HL60 cells ERK1/2 is activated in the early phase of 1,25D-induced differentiation, and this can be accentuated by serum starving the cells prior to exposure to 1,25D (Marcinkowska et al, 1997;Marcinkowska, 2001;Wang and Studzinski, 2001a).…”

Signal transduction pathways that contribute to myeloid differentiation

“…232 Activation of the PI3K/Akt pathway was also observed during 1,25-dihydroxyvitamin D3-induced monocytic differentiation of HL-60 cells. 233 Inhibition of the Akt pathway with pharmacologic inhibitors enhanced vitamin D3-induced cell cycle arrest, increased the expression of p27 Kip1 and promoted apoptosis in HL-60 cells. 233 Inactivation of PTEN (phosphatase and tensin homologue), a negative regulator of the PI3K/Akt pathway, results in expansion of bone marrow hematopoietic stem cells.…”

“…233 Inhibition of the Akt pathway with pharmacologic inhibitors enhanced vitamin D3-induced cell cycle arrest, increased the expression of p27 Kip1 and promoted apoptosis in HL-60 cells. 233 Inactivation of PTEN (phosphatase and tensin homologue), a negative regulator of the PI3K/Akt pathway, results in expansion of bone marrow hematopoietic stem cells. 234 Moreover, mice with a mutant PTEN develop conditions reminiscent of AML or lymphoid leukemia.…”