In order to improve the therapeutic index of conventional anticancer drugs without inducing global toxicity in patients, we set up this study to investigate the enhancing effect of TPL for two antileukemia agents in drug-resistant myeloid leukemia cell lines. Mechanism of how TPL enhances the drug-sensitivity of resistant myeloid leukemia cell lines was also explored in our study to provide an experimental base to elucidate how TPL might affect the treatment outcome of patients with high levels of certain genes.
During recent years, TPL has been proven to have anticancer properties through its marvelous antiproliferative activity and induction of apoptosis in a variety of cancers in vitro and in vivo.[4,6–8] Although use of TPL as a single agent for cancer indication is limited by its nonspecific toxicity and low therapeutic index, it may be used at a relatively low dosage to enhance the cytotoxicity of other chemotherapy drugs.[9,11–13] Our study also demonstrates that TPL is highly toxic to the DOX-resistant leukemia cell line HL60/A and the IM resistant cell line K562/G in vitro in a dose-dependent manner. Low concentrations of TPL significantly enhance the cytotoxicity of DOX in HL60/A and IM in K562/G by 1.82- and 1.18-fold, respectively. The enhancement is more powerful when DOX or IM is used at a higher concentration. CI analysis also showed that TPL and anticancer agents have synergistic effects when the fraction affected is below 60% (DOX) and 90% (IM). Our results also indicate that TPL enhances DOX-induced and IM-induced apoptosis in HL60/A and K562/G cells. Compared with DOX- or IM-treated cells, flow cytometric analysis shows that after 24 h exposure with anticancer drugs and TPL, the apoptotic cell population is significantly increased in HL60/A cells (19.55 ± 1.70% vs 72.62 ± 4.83%, 3.71-fold, p < 0.01) and K562/G cells (24.78 ± 1.12% vs 77.52 ± 7.75%, 3.12-fold, p < 0.01).
HIF-1α is the major mediator of hypoxic responses in malignant cells. It can induce multiple target genes such as VEGF (an important angiogenesis factor),CXCR4 (an important factor regulating cell migration),BNIP3 (a pro-cell death member of the Bcl-2 family) and CAIX (a marker for hypoxia). Thus, HIF-1α can activate downstream pathways involved in proliferation, metabolism, differentiation and angiogenesis.[34,35]
Resistant cells usually express more HIF-1α. Zhao et al. found that IM-resistant cell lines expressed more HIF-1α than sensitive cell lines. Our study also demonstrated elevated HIF-1α mRNA levels in resistant cell lines than their parental cells (Figure 1). Moreover, genetic modulation of HIF-1α might also occur in resistant cells. Usually, HIF-1α expression could not be detected under normoxia conditions at the protein level in HL60 cells. However, in our study, expression of HIF-1α could be observed under normoxia conditions in resistant HL60/A cell lines. Wang et al. have reported that HIF-1α protein was selectively enriched in the CD38−CD34+ subsets (acute myeloid leukemia stem cell marker) in comparison to in the CD38−CD34− subset. One major characteristic of acute myeloid leukemia stem cells is drug resistance. It is reasonable to hypothesize that resistant cells might contain a greater proportion of stem cells than sensitive cells with genetically modulation of HIF-1α.
Furthermore, genetic modulation of HIF-1α could lead to the change of IM and DOX ability. Doublier et al. found that transfecting cells with specific siRNA for HIF-1α significantly increased the intracellular DOX accumulation in MCF7 3D spheroids. Zhao et al. found that after 10 days of culture with medium containing IM, few cells survived in the cultures transfected with the HIF-1α shRNA expression plasmid, while cultures transfected with the control shRNA had undergone a fivefold expansion. All these publications demonstrate a close relationship between HIF-1α and drug sensitivity.
Owing to the important role of HIF-1α in drug resistance, we explored whether TPL plus anticancer agents could affect the function and expression of HIF-1α. A previous study has reported on the ability of TPL to inhibit HIF-1. HIF-1α is an important component of HIF-1, in accordance with the previous study, our results also showed the downregulation of HIF-1α at both the mRNA and protein levels, and downstream genes, for example, BNIP3, VEGF and CAIX at the mRNA level and CXCR4 at the protein level (Figures 4 & 5A & C). However, there is one publication showing that TPL increases the levels of HIF-1α mRNA and protein and reduces HIF-1α transcriptional activity in SKOV-3 cells after 12 h exposure to different concentrations of TPL. This might be because of the longer exposure time in our study (12 vs 24 h) and different cell type (SKOV-3 vs K562/G and HL60/A) in different experiments. The elevated HIF-1α mRNA levels in the previous study might be a compensatory response by the tumor cells in an effort to maintain HIF-1α transcriptional activity to protect cells from apoptosis, while after longer exposure time, cells might be unable to maintain homeostasis and thus the apoptotic program was activated and protective factors such as HIF-1α were decreased to induce apoptosis.
The transcription factor Nrf2 is a potent transcriptional activator and plays a central role in the protection of cells against oxidative damage. It can regulate a lot of downstream genes with a wide variety of functions, such as cellular redox homeostasis, cell growth and apoptosis, DNA repair, the inflammatory response and the ubiquitin-mediated degradation pathway.
It has already been demonstrated that high basal nuclear levels of Nrf2 in leukemia cells could reduce sensitivity to proteasome inhibitors. Zhong, et al. have found that the MCF-7/DOX cell line expressed more Nrf2 mRNA than its parental cell line. Tarumoto et al. reported that sensitivity to IM could be restored by suppression of Nrf2-dependent genes while Nagai et al. also found that Nrf2 is essential in the hemin-mediated decrease in IM sensitivity. Our study also found elevated Nrf2 mRNA levels in resistant cell lines compared to their parental cell lines, further indicating that elevation of Nrf2 might lead to the treatment failure (Figure 1). Downregulation of Nrf2 in many cancer cells by drugs could also lead to the enhancement of sensitivity. Ren et al. found that inhibition of the Nrf2 pathway by brusatol could sensitize a broad spectrum of cancer cells and A549 xenografts to cisplatin and other chemotherapeutic drugs.
Moreover, genetic modulation of Nrf2 could affect the ability of DOX and IM. Zhong et al. found that genetic knockdown of endogenous Nrf2 sensitizes MCF-7 cells to DOX. While Nagai et al. found that knockdown of Nrf2 expression by RNAi largely abolished the effect of hemin in reducing the cellular sensitivity to IM. All these publications demonstrated a key role for Nrf2 in drug sensitivity.
Thus we explore whether the ability of TPL in enhancing drug sensitivity is correlated with the TPL-induced change of Nrf2 expression and function. A previous study shows that expression of Nrf2 in rat kidney cells decreased below basal level after 24 h exposure to TPL although the expression of Nrf might elevate if cells were treated with TPL for a shorter period. Our study also reveals that TPL could significantly downregulate Nrf2 expression both at the mRNA and protein levels after HL60/A and K562/G cells were exposed to different treatment for 24 h, with the highest downregulation occurring in the TPL plus antiagents group (Figures 4 & 5B & D). This phenomenon indicated that TPL-induced cytotoxicity might overcome adaptive Nrf2 protection for cells. Moreover, downregulation of downstream genes of Nrf2 such as xenobiotic-metabolizing enzyme, NQO1,GSR, as well as the antioxidative enzyme, HO-1 was also observed in our study (Figure 5B & D). These target genes of Nrf2 can form a network of reactions, resulting in protection against oxidative insults and in enhanced cell survival.[46,47] Downregulation of mRNA level of these genes in TPL-treated cells was also observed in our study (Figure 5B & D), with the highest downregulation occurring in TPL plus antiagent group, further indicating that TPL might induce cell apoptosis through inhibition of Nrf2 pathway. Next, we will further look into the ability of TPL to enhance the drug sensitivity of relapse patients who have higher levels of Nrf2, providing the basis for more individualized therapy.
Pharmacogenomics. 2013;14(11):1305-1317. © 2013 Future Medicine Ltd.