1-[2-(2-Methoxyphenylamino)ethylamino]-3-(naphthalene-1-yloxy)propan-2-ol May Be a Promising Anticancer Drug

We have originally synthesized the naftopidil analogue 1-[2-(2-methoxyphenylamino)ethylamino]-3-(naphthalene-1-yloxy)propan-2-ol (HUHS 1015) as a new anticancer drug. HUHS1015 induces cell death in a wide variety of human cancer cell lines originated from malignant pleural mesothelioma, lung cancer, hepatoma, gastric cancer, colorectal cancer, bladder cancer, prostate cancer, and renal cancer. HUHS1015-induced cell death includes necrosis (necroptosis) and apoptosis, and the underlying mechanism differs depending upon cancer cell types. HUHS1015 effectively suppresses tumor growth in mice inoculated with NCI-H2052, MKN45, or CW2 cells, with a potential similar to or higher than that of currently used anticancer drugs. Here we show how HUHS1015 might offer brilliant hope for cancer therapy.


Introduction
Naftopidil, an antagonist for the α1-adrenoceptor, with high selectivity for α1A-and α1D-receptors, has been clinically used as a drug for treatment of benign prostate hyperplasia and hypertension [1]. Accumulating evidence has shown that naftopidil exhibits an anticancer effect. Naftopidil inhibits prostate cancer cell growth by arresting them at the G1 phase of cell cycling [2,3]. In our studies, naftopidil induced cell death for bladder, prostate, renal cancer, and malignant pleural mesothelioma (MPM) cell lines [4,5]. The mechanism for the anticancer action of naftopidil remains to be explored. α1-Adrenoceptor is divided into α1A-, α1B-, and α1D-subtypes, and the receptor is linked to Gq/11 protein-bearing phospholipase C activation followed by protein kinase C (PKC) activation [6][7][8]. Therefore, one would speculate that naftopidil should suppress PKC activation by inhibiting the α1-adrenoceptor. Surprisingly, the PKC inhibitor GF109203X attenuated naftopidil-induced apoptosis of MPM cells [5]. Moreover, knocking down the α1D-adrenoceptor promoted proliferation of MPM cells [5]. Collectively, these data suggest that naftopidil induces MPM cell death by a mechanism independent of α1-adrenoceptor blocking.
We have synthesized 21 naftopidil analogues ( Figure 1) and assessed the anticancer effect of each compound.
Caspase-8 is activated through death receptors. The death receptor Fas, activated by FasL, recruits the adaptor protein FADD to aggregate procaspase-8, which cleaves to one other to initiate an active form of caspase-8 and, in turn, to activate the effector caspase-3 [16]. TNFα activates TNFR1, which forms a complex of TRADD/FADD/procaspase-8 to activate caspase-8 followed by the effector caspase-3 [17]. Collectively, HUHS1015 could activate caspase-8 by activating TNFR1 in association with upregulation of TNFα expression, followed by activation of the effector caspase-3, to induce apoptosis.

HUHS1015 Induces Caspase-Independent Apoptosis by Accumulating AMID in the Nucleus
HUHS1015 significantly increased nuclear localization of AMID in parallel with decreased cytosolic localization in MKN28 cells [18]. An increase in the nuclear localization of AIF was not obtained with HUHS1015, but, conversely, nuclear localization of AIF was decreased [18]. HUHS1015 had no effect on expression of mRNAs and proteins for AIF and AMID in MKN28 cells [18].
In response to lethal signals, AIF is translocated from the mitochondria into the nucleus, where it binds to the nuclear DNA, thereby causing chromosomal condensation, margination, and large-scale DNA fragmentation (approximately 50-kb fragments) [19,20]. The AIF homologue AMID is identified as a human pro-apoptotic protein and designated as p53-responsive gene 3 [21][22][23][24][25][26]. AMID is preferentially localized in the outer mitochondrial membrane or the cytoplasm. In response to apoptotic stimuli, AMID is translocated in the nucleus and non-specifically binds to DNAs to induce DNA fragmentation, i.e., apoptosis [23][24][25][26]. Taken together, HUHS1015 induces caspase-independent apoptosis by accumulating AMID in the nucleus.

HUHS1015 Induces Necrosis (Necroptosis) of Cancer Cells
In the flow cytometry using propidium iodide (PI) and annexin V, PI is a marker of dead cells and annexin V, which detects externalized phosphatidylserine residues, is a marker of apoptotic cells [27]. In this assay, each population of PI-positive/annexin V-negative, PI-negative/annexin V-positive, or PI-positive/annexin V-positive cells corresponds to primary necrosis, early apoptosis, and late apoptosis/secondary necrosis, respectively [28].
Cell death is classified into three types: apoptosis, necrosis, and necroptosis. Necroptosis is regarded as an alternative form of programmed cell death [29]. Death receptors such as TNFα receptor and Fas or pro-apoptotic Bcl-2 family members related to mitochondrial damage mediate both in apoptosis and necroptosis. RIP1 associated with death receptors forms complex IIa including FADD and caspase-8, causing activation of caspase-8 followed by caspase-3 [30]. In a different pathway, caspase-8 proteolyzes Bid into truncated Bid (tBid), which makes pores in the mitochondria, allowing release of apoptosis-related factors involving activation of caspase-9 and the effector caspase-3 [31]. RIP1 is phosphorylated by RIP1 kinase and forms complex IIb, together with RIP3, to induce necroptosis [30]. Apoptotic stimuli such as oxidative stress cause pro-apoptotic Bcl-2 family member-mediated damage of mitochondria, thereby releasing cytochrome c to activate caspase-3/9 and induce mitochondrial apoptosis [32]. Mitochondrial damage, alternatively, activates ATPase and reduces intracellular ATP concentrations to induce necroptosis [33]. Overall, HUHS1015 is likely to induce both necrosis (necroptosis) and apoptosis of cancer cells.

HUHS1015 Suppresses Tumor Growth in Xenograft Model Mice
HUHS1015 clearly suppressed tumor growth in mice inoculated with NCI-H2052 cells, with a potential much greater than that for paclitaxel [10]. The survival rate at two months after HUHS1015 treatment was 100%, but it was only 30% for paclitaxel-treated mice [10]. In mice inoculated with MKN45 cells, HUHS1015 obviously suppressed tumor growth, but naftopidil otherwise had no significant effect. This indicates that the naftopidil analogue HUHS1015 is more effective on gastric cancer cells than naftopidil by itself. The anticancer drugs cisplatin, paclitaxel, and irinotecan also suppressed tumor growth, and the order of the potential for all the investigated compounds was irinotecan >> HUHS1015≒ cisplatin > paclitaxel > naftopidil. The survival rate at 33 days' treatment with HUHS1015 was 100%, while that for cisplatin, irinotecan, and paclitaxel treatment was 43%, 71%, and 86%, respectively. HUHS1015 also inhibited tumor growth in mice inoculated with CW2 cells, with the survival rate being 100% at 33 days' treatment. In addition, no remarkable body weight loss was found throughout experiments and the blood examination exhibited no liver and renal dysfunction. Overall, these data show that HUHS1015 serves as a beneficial anticancer drug, with lesser side effects.

Conclusions
The naftopidil analogue HUHS1015 induces both necrosis (necroptosis) and apoptosis in a wide variety of cancer cells. The former may be caused by intracellular ATP reduction in association with mitochondrial damage or activation of the RIP1 kinase ( Figure 4). The latter may be due to caspase activation through the mitochondria and TNFα receptor for caspase-dependent apoptosis and to AMID accumulation in the nucleus for caspase-independent apoptosis (Figure 4). HUHS1015 effectively prevented tumor growth in the xenograft model mice, with lesser side effects. HUHS1015 could, thus, be developed as a new type of promising anticancer drug.

Author Contributions
Tadashi Shimizu and Akito Tanaka synthesized naftopidil analogues. Takeshi Kanno, Ayako Tsuchiya, and Yoshiko Kaku designed and performed experiments, and analyzed data. Tomouki Nishizaki conducted the experiments and wrote the manuscript. All the authors approved the manuscript.