The Antileukemia Activity of Natural Product HQ17(3) Is Possibly Associated with Downregulation of miR-17-92 Cluster

The compound 10′(Z),13′(E),15′(E)-heptadecatrienylhydroquinone [HQ17(3)] was purified from the sap of the lacquer tree Rhus succedanea. HQ17(3) has cytotoxic effect on cancer cells and can inhibit topoisomerase (topo) IIα activity. We treated various cancer cells with different doses of HQ17(3) and found that leukemia cells were most sensitive to HQ17(3). After analysis of microRNA (miRNA) profiling, we found that treatment with HQ17(3) caused downregulation of miR-17-92 cluster in some leukemia cells. These changes partially restored the normal levels from leukemia-specific miRNA expression signature. Messenger RNAs of tumor suppressor proteins, such as pRB, PTEN, and Dicer, are targets of miR-17-92 cluster. Their protein levels were increased after the treatment. c-Myc is a regulatory protein for miR-17-92 gene. Similar to topo IIα, we found that c-Myc decreased its activity after the HQ17(3) treatment, which may explain the downregulation of miR-17-92 cluster. Combined with 5-fluorouracil, NaAsO2, or ABT-737, HQ17(3) elicited additive inhibitory effects on leukemia cells. In conclusion, the high sensitivity of leukemia cells to HQ17(3) may be associated with the reduction of topo IIα and c-Myc activities, as well as with the downregulation of the miR-17-92 cluster expression.


Introduction
The compound 10 (Z),13 (E),15 (E)-heptadecatrienylhydroquinone [HQ17 (3)] is a new natural product purified from the sap of the lacquer tree Rhus succedanea [1]. HQ17(3) is composed of 1.5% to 2% dry weight. HQ17(3) effectively and irreversibly inhibits topoisomerase (topo) II activity by reacting with some cysteine residues of this enzyme [2]. A cell-based assay has shown that HQ17(3) inhibits the growth of topo II-deficient cells HL-60/MX2 with an EC50 of 9.6 M and exerts no effect on peripheral blood mononuclear cells at concentrations of up to 50 M [3]. Therefore, HQ17(3) attacks other targets on leukemia cells;, so obtaining more information about this drug is worthwhile.
The miR-17-92 polycistron promotes several aspects of oncogenic transformation, including evasion of apoptosis [14]. Further dissection of the miRNA components in this cluster reveals that the miR-17/20a seed family accounts for antisenescence activity, with targets such as pRB and 2 BioMed Research International E2F1 [15]. The miR-19a/b inhibits apoptosis by suppressing PTEN (a tumor suppressor protein) [16,17]. The 3 UTR of Dicer messenger RNA (mRNA) contains two miR-18a binding sites. Dicer cleaves pre-miRNA to produce active miRNA duplex; it functions as an antiproliferation protein in general [18,19]. Taken together, the members of miR-17-92 cluster promote tumorigenesis by antagonizing senescence, apoptosis, and tumor-suppressing mechanisms.
In this study, we found that treatment with HQ17(3) caused a decrease of c-Myc activity and downregulation of miR-17-92 clusters in some leukemia cells. This finding may explain why leukemia cells were selectively sensitive to this compound. HQ17(3) possibly has a significant role in antileukemia treatment. (3). The purification steps for HQ17(3) are the same as those described in our previous study [20]. HQ17(3) was dissolved in 50% alcohol and kept at −20 ∘ C. The working concentrations of HQ17(3) were 1, 3, and 9 mM. Therefore, cells treated with 1, 3, or 9 M of HQ17(3) can have the same concentration of alcohol. For blank control, the same amount of 50% alcohol was added.

Microarray.
The miRNAs from these cells were isolated by TRIzol (Invitrogen, Carlsbad, CA, USA) according to the protocol provided by the manufacturer. After passing the standard quality control, these miRNA levels were quantified by Agilent human miRNA array R12 and GeneSpring GX software. miRNA profiles were also measured by NanoString nCounter miRNA (NanoString Technologies, Inc., Seattle, WA, USA) which are based on direct digital detection of mRNA molecules using color-coded probes without the sequence amplification step [21].

Detection of miRNA Levels through qRT-PCR Analysis.
The K562, Ramos, and Molt-4 cells were treated with 3 M   bands were revealed by ECL system (Perkin Elmer, Inc., Boston, MA, USA) and developed on X-ray films.  Assay System and Ready-To-Glow Reporter Assay (Clontech, Inc., Palo Alto, CA, USA), respectively, according to the manufacturer's instruction.  Figure 1. HQ17(3). Aberrant expression of specific miRNAs has recently been addressed for CLL and other B-cell lymphomas [8]. Therefore, we monitored the change in miRNA profile after treatment with HQ17(3). U937 cells were treated with 3 M of HQ17(3) for 24 h. The changes in miRNA levels were detected by Agilent human miRNA array R12. The miRNA levels that showed apparent change (>1.43-fold or <0.7-fold and signal >60) were screened and presented in Figure 2(a). We found that the downregulated miRNAs, that is, miR-17, miR-18a, miR-19a/b, miR-20a, and miR-92a, all belong to a polycistron known as miR-17-92 cluster. We also measured miRNA profiles by NanoString nCounter miRNA. The treated Ramos cells (3 M, 24 h) showed different extents of downregulation of miR-17 (0.78), miR-18a (0.52), miR-19a (0.59), and miR-20a (0.92). Moreover, qRT-PCR was used to verify level change of miR-17 and miR-19a in K-562, Molt-4, and Ramos cells. The results are shown in Figure 2(b). Decrease in miR-17 and miR-19a was observed in these HQ17(3)-treated cells. We speculated that the downregulation of miRNA in miR-17-92 cluster after treatment with HQ17(3) may be a general phenomenon for leukemia cells. by HQ17(3). Some mRNAs of tumor suppressor proteins, such as pRB, PTEN, and Dicer, are the targets for miRNA members of miR-17-92 cluster [15][16][17]. Therefore, the downregulation of miR-17-92 may cause an increase of these proteins. Western blot analyses were conducted. The results are shown in Figure 3. Protein levels of pRB, PTEN, and Dicer elevated to some extent for leukemia cells treated with HQ17(3). reacts and modifies some cysteine residues of topo II in vitro and in cells [2]. However, Cys-427 modification was found only in the cellular system [2]. HQ17(3) also possibly reacts and modifies cysteine residues of c-Myc. Therefore, we created amino acid sequence alignments of c-Myc and topo II using the website http://blast.ncbi.nlm.nih.gov/Blast .cgi?CMD=Web&PAGE TYPE=BlastHome. Interestingly, a consensus-like sequence was found close to Cys-257 of c-Myc and Cys-427 of topo II (Figure 4(b)). This consensus-like sequence may be a hot spot for the attack of HQ17(3). (3). Finally, we tried to elucidate any additive or synergistic effect when HQ17(3) is combined with other anticancer drugs. 5-FU, NaAsO 2 , and ABT-737 were tested. Leukemia cells Molt-4 and Ramos were treated with different doses of anticancer drug with or without 3 M of HQ17(3). The results are shown in Figure 5. Although synergistic effect was not found, an additive effect was prominent when HQ17(3) and the aforementioned anticancer drugs were used together. Specifically, HQ17(3) exerted no toxic effect on normal peripheral blood mononuclear cells at concentrations of up to 50 M [3]. In conclusion, the high sensitivity of leukemia cells to HQ17(3) may be associated with the reduction of topo II and c-Myc activities, as well as with the downregulation of the miR-17-92 cluster expression. Natural product HQ17(3) itself may be an anticancer drug or have a significant function in sensitizing leukemia cells to anticancer drugs.