In the treatment of APL, the successful use of arsenic compounds contained in some traditional Chinese remedies revealed the potential of TCM in cancer therapy [3
]. We used the APL cells as a model to evaluate the anticancer effects of another Chinese remedy THL, which is a herbal mixture consisting mainly of extracts from 14 Chinese medicinal herbs and has been used as an anticancer dietary supplement for more than 10 years. In this study, we comprehensively elucidated the molecular anticancer mechanisms of THL against APL cells in the aspects of apoptosis induction, cell-cycle regulation, down-regulation of PML-RARα
fusion protein and suppression of oncogenic signaling pathways.
Cell-cycle progression is closely related to the proliferation of cancer cells and therefore becomes the reasonable target for cancer therapy [5
]. There are several types of agents targeting the cell-cycle regulating proteins to arrest the cell cycle including chemotherapeutics. Chemotherapy agents such as taxol [27
] and etoposide [28
] induced G2/M arrest in cancer cells accompanied with the increase of cyclin B1. Similarly, ATO increased cyclin B1 in NB4 APL [7
] and MCF-7 breast cancer [29
] cells but decreased cyclin B1 in PCI-1 head and neck cancer cells during G2/M arrest induction [30
]. In THL-induced G2/M arrest of NB4 leukemia cells, the cyclin B1 was also decreased rather than increased. This phenomenon was frequently observed in cancer cells treated with natural anticancer products, such as Huanglian
], theaflavin (black tea polyphenol) [32
], and genistein [33
]. As THL is composed of a mixture of Chinese herbal extracts, it would be expected to decrease cyclin B1 during G2/M arrest induction.
THL decreased not only the cyclin B1 but also the cyclin A protein level of NB4 cells. This phenomenon was also seen in the G2/M arrest induced by other herbal extracts or phytochemicals, such as Physalis angulata
] and carnosic acid, a polyphenol present in Rosmarinus officinalis
]. In THL-treated NB4 cells, the cyclin A was suppressed by a smaller dose than cyclin B1. This phenomenon was similar to that induced by carnosic acid in Caco-2 colon cancer cells [34
], which resulted in arresting the cell cycle prior to prometaphase. Therefore, it is speculated that the major cell-cycle arrest induced by THL may occur before prometaphase.
A hallmark of APL is a chromosomal translocation that results in the formation of PML-RARα
fusion protein, which blocks both the apoptosis and differentiation of APL cells [8
]. According to our findings, THL induced dramatic apoptosis and down-regulation of PML-RARα
fusion protein in NB4 cells but no differentiation induction was observed (data not shown). Nason-Burchenal et al. [35
] had demonstrated that cleavage of PML-RARα
mRNA by using a hammerhead ribozyme could trigger only apoptosis in NB4 APL cells but was insufficient to induce differentiation. Therefore, it is possible that THL may target the PML-RARα
mRNA by the similar manner, which remains to be explored.
Recent research has shown that the oncogenic effects of PML-RARα
fusion protein in leukemia were closely related to the hypermethylation on the promoters of target genes by recruiting DNMT [36
]. DNMT1, the most important DNMT for maintaining the aberrant methylation [14
], had been found to be aberrantly elevated not only in leukemia [9
] but also in many types of cancer cells [37
]. THL dramatically decreased both PML-RARα
fusion protein and DNMT1 during apoptosis induction. The decrease of DNMT1 may play an important role in THL-induced apoptosis of NB4 cells as it is reported that depletion of DNMT1 could induce or promote apoptosis of cancer cells [43
]. Inhibition of DNMT1 had been suggested as one of the targeted therapies for hematologic malignancies [45
]. How to down-regulate DNMT1 appears as a sound approach for epigenetic cancer therapies [14
]. Considering its DNMT1 suppressing effect, THL might have potential as a candidate agent for epigenetic cancer therapy.
THL-induced apoptosis of NB4 cells may closely correlate with the inhibition of the oncogenic signaling pathways, such as Akt/mTOR, Stat3, and ERK. In acute leukemia cells, the Akt signaling is frequently activated and strongly contributes to proliferation and survival [21
]. Constitutive Stat3 activation has been observed in a number of hematopoietic malignancies, and the apoptosis resistance in APL cells was hypothesized to be mediated through the augmentation of Stat3 signaling by the APL fusion proteins including the PML-RARα
]. The ERK signaling is frequently activated in AML cells and down-regulation of this aberrantly activated ERK had been shown to result in significant apoptosis of the AML blasts [20
]. As targeting these three signaling pathways for effective leukemia therapy has been suggested [15
], the inhibition of all these three cascades by THL further reveals its potentiality on APL therapy. Furthermore, the inhibitory effect of THL on ERK phosphorylation is notably dramatic. This may be useful for enhancing the therapeutic efficacy of another APL treating agent ATO. The ATO-induced apoptosis of acute leukemia cells is counteracted by the induction of ERK activity [50
], and inhibition of ERK activity could enhance apoptosis in ATO-treated NB4 cells [51
]. Therefore, it is speculated that combined or sequential use with THL plus ATO may provide the beneficial for APL treatment.
In addition to elucidate the comprehensive effects of THL against APL cells, we also purified the active fraction EAS5 from this herbal mixture. It seems that the active anticancer components in THL are relative hydrophobic, because the EAS5 was ethyl acetate soluble and eluted by ethyl acetate/n-hexane (80/20). This hydrophobic property of EAS5 may make it easier to be absorbed by GI tract. As the EAS5 was about one hundred times more potent than THL and exerted most of its multi-targeting effects against NB4 cells, it may be considered as a candidate for future clinical trial of complementary APL treatment.