KML001 Induces Apoptosis and Autophagic Cell Death in Prostate Cancer Cells via Oxidative Stress Pathway

We investigated the effects of KML001 (NaAsO2, sodium metaarsenite, Kominox), an orally bioavailable arsenic compound, on the growth and death of human prostate cancer cells and its mechanism of action. Growth inhibition was assessed by cytotoxicity assays in the presence or absence of inhibitor of apoptosis, inhibitor of autophagy or antioxidant N-Acetyl-L-cysteine to study mechanism of cell death induced by KML001 in PC3, DU145 and LNCaP prostate cancer cell lines. Electron microscopy, flow cytometry and Western blotting were used to study apoptotic and autophagic mechanisms. The DU145 xenograft model was used to determine the efficacy of KML001 in vivo. KML001 decreased the viability of cells and increased the percentage of annexin V-positive cells dose-dependently in prostate cancer cells, and LNCaP cells were more sensitive to KML001 than PC3 or DU145 cells. Electron microscopy revealed typical apoptotic characters and autophagic vacuoles in cells treated with KML001. Exposure to KML001 in prostate cancer cells induced apoptosis and autophagy in a time- and dose-dependent manner. KML001 induced dose-dependent accumulation of reactive oxygen species, and scavenging the reactive oxygen species with N-Acetyl-L-cysteine reduced LC3 and cleaved poly (ADP-ribose) polymerase. KML001 significantly inhibited tumor growth in the DU145 xenograft model. In addition, significant decrease of proliferation and significant increases of apoptosis and autophagy were observed in KML001-treated tumors than in vehicle-treated tumors. Exposure of human prostate cancer cells to KML001 induced both apoptosis and autophagic cell death via oxidative stress pathway. And KML001 had an antiproliferative effect on DU145 cells in xenograft mice.


Introduction
While arsenic compounds are widely known as carcinogens that induce cancers in many human tissues, arsenic trioxide (As 2 O 3 , ATO) has been demonstrated clinically to be an effective therapeutic agent for the treatment of acute promyelocytic leukemia [1,2]. Although its anticancer mechanism of action is not well understood, ATO has been found to regulate various biological functions, including cell proliferation, apoptosis, differentiation, and angiogenesis in various cell lines [3]. Because ATO was successful in treating acute promyelocytic leukemia [1,2,4], arsenicals are experiencing a revival in modern cancer medicine [5].
Due to its oral bioavailability, water solubility and lower median lethal dose (LD 50 ) in rats, KML001 is more suitable for clinical applications than ATO [13]. We therefore investigated the ability of KML001 to inhibit the growth and induce cell death of human prostate cancer cell lines. We also analyzed whether autophagy is with apoptosis involved in KML001-induced cell death in prostate cancer cell lines. Finally, we determined the antitumor effect of KML001 in DU145 xenograft model.

Assays for apoptosis detection
Human prostate cancer cells were exposed to KML001 and apoptosis was assessed by flow cytometry using the annexin V-FITC Apoptosis Detection Kit (BD Biosciences, Bedford, MA, USA) according to the manufacturer's instructions, and analyzed as described previously [15].

Western blot analysis
Human prostate cancer cells cultured in RPMI 1640 with 5% heat inactivated FBS were treated with KML001 at various concentrations for different time periods, followed by incubation for 6 h with and without Z-VAD-FMK (20 μM) or 3-MA (2 mM). Western blot analysis was performed as described previously [15]. Antibodies to LC3 (Novus Biologicals, Littleton, CO, USA), procaspase-3, and poly (ADP-ribose) polymerase (PARP, Cell Signaling Technology, Danvers, MA, USA), and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for Western blot analysis.

DU145 xenograft animal model
All aspects of animal care and treatment were performed according to the eighth edition of the Guide for the Care and Use of Laboratory Animals published in 2011. The protocol was approved by Institutional Animal Care and Use Committee of Asan Medical Center, Seoul, Korea (2012-02-067). Four-week-old male BALB/C nude mice (OrientBio, Seoul, Korea) were subcutaneously inoculated with 5 × 10 6 DU145 cells. When the tumors reached an average volume of about 100 mm 3 , the mice were randomly divided into control and treatment groups (6 animals per group). For the DU145 bearing mice, the treatment groups were administered KML001 (2.5 or 10 mg/kg/d) and daily by oral gavages for 4 weeks. Mice were monitored for toxicity by body weight measurements, and tumors were measured three times a week and volume was calculated by the modified ellipsoid formula: 0.52×length×(width) 2 [17]. Mice were euthanized by carbon dioxide after harvesting tumors.

TUNEL assay and Ki-67 immunohistochemical staining
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) for the detection of apoptotic cells were performed using the in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's protocols. For image analysis, three randomly selected fields from each mice were photographed at 200× magnification using fluorescence microscopy (Carl Zeiss, Jena, Germany).
For Ki-67 immunostaining using proliferation markers, tissue paraffin-embedded sections were deparaffinized using xylenes and graded ethanol followed by antigen retrieval using IHC-Tek epitope retrieval steamer set (IHC World, LLC. Woodstock, MD, USA). Slides staining were performed as described previously [18]. For images and intensity scoring, three randomly selected fields from each mice were photographed at 200× magnification using an Olympus U-LH100L-3 camera and Olympus Ix 71 software.

Statistical Analysis
All data were shown as the means ± standard deviations (SD). Statistical significance was considered at p < 0.05 and determined by one-way analysis of variance (ANOVA).

Effect of KML001 on proliferation of prostate cancer cells
Incubation of androgen-independent PC3 and DU145 and androgen-dependent LNCaP prostate cancer cells with 0.0001-200 μM KML001 for 72 h reduced cell viability in a dose-dependent manner. LNCaP cells were more sensitive than PC3 or DU145 cells (Fig 1; Table 1).

Induction of apoptotic cell death
To determine whether treatment with KML001 (10 μM) induces apoptosis, the ultrastructure of KML001-treated PC3 cells was analyzed by electron microscopy. PC3 cells treated with KML001 showed typical apoptotic characters, including a smaller nucleus, more concentrated cytoplasm, crumpled nuclear membrane, and chromosomes condensed into a semilunar shape with attachments to the nuclear and cellular membranes. Moreover, apoptotic bodies were observed when the chromatin condensed and ruptured to the nuclear margin. Similar results were observed in DU145 and LNCaP cells treated with IC 50 concentrations of KML001 (5 μM and 1 μM, respectively) (Fig 2).  To investigate the mechanism of KML001-induced cell death, we performed annexin V flow cytometry assays. Treatment of these cells with KML001 yielded cells positive for annexin V staining only and cells positive for annexin V and PI staining. These results indicate that KML001 induced cell death via both apoptosis and necrosis (Fig 3A).
We also found that exposure of prostate cancer cells to KML001 resulted in a time-and dose-dependent increase in the cleavage of PARP, as well as the activation of procaspases-3, which mediates intrinsic apoptosis (Fig 3B). To confirm the role of apoptosis in the KML001-induced death of prostate cancer cells, we treated the cells with the caspase inhibitor Z-VAD-FMK. We found that addition of 20 μM Z-VAD-FMK decreased the activation of procaspases-3 in all prostate cancer cell lines (data not shown), supporting the hypothesis that KML001 induced apoptosis in prostate cancer cells.

Induction of autophagic cell death
To determine whether treatment with KML001 (10 μM) induces autophagy, the ultrastructure of PC3 treated cells was examined by electron microscopy. We observed autophagic vacuoles, including autophagosomes or autolysosomes, in KML001-treated PC3 cells, as well as in DU145 and LNCaP cells treated with their respective IC 50 concentrations of KML001 (Fig 2).
Prostate cancer cells exposed to KML001 were examined by Western blotting, using an anti-LC3 antibody that recognizes both forms of LC3. We found that expression of LC3 and/or conversion to LC3-II increased in a time-and dose-dependent manner, relative to untreated cells (Fig 4A). To confirm the role of autophagy in the KML001-induced death of prostate cancer cells, we treated the cells with the autophagy inhibitor 3-MA. We found that addition of 2 mM 3-MA decreased the expression of LC3-II in all prostate cancer cell lines (Fig 4B). In addition, addition of 1 mM 3-MA also attenuated KML-induced cell death in all prostate cancer cell lines (Fig 4C). Taken together, these findings support hypothesis that KML001 induced autophagy-mediated cell death in prostate cancer cells.

The antioxidant NAC protects cells from KML001-induced cell death
KML001 induced dose-dependent ROS accumulation in all 3 prostate cancer cell lines ( Fig  5A). We also examined the effects of the antioxidant NAC on KML001-induced apoptosis and autophagy. We found that treatment of all 3 prostate cancer cell lines with 1 mM NAC decreased the expression of LC3-II and the proteolysis of PARP (Fig 5B). In addition, addition  of 1 mM NAC also attenuated KML-induced cell death in all prostate cancer cell lines (Fig 5C). These findings provide further evidence that exposure of prostate cancer cells to KML001 activated both apoptosis and autophagy via oxidative stress.

Effect on DU145 xenografts
We subcutaneously injected nude mice with 5 × 10 6 DU145 cells to test the effects of KML001 on androgen-independent prostate cancer cells in vivo. After the tumor size reached 100 mm 3 , we administered KML001 (2.5 or 10 mg/kg/d) to them for 4 weeks. In the DU145 xenograft model, tumor growth was inhibited with KML001 as compared to vehicle (Fig 6A). But KML001 treatment had no effect on body weight of mice (Fig 6B)   In the next experiment, we determined the effect of KML001 on proliferation, apoptosis, and autophagy in tumor tissues harvested from animals of different treatment groups. In immunohistochemical staining of the proliferation marker Ki-67, significant decreases in Ki-67 immunostaining were observed in KML001-treated tumors than in vehicle-treated tumors (Fig 7A). In TUNEL assay, significant greater apoptotic cells were observed in KML001-treated tumors than in vehicle-treated tumors (Fig 7B). In Western blot analysis, we found that expression of LC3 and/or conversion to LC3-II increased in a dose-dependent manner, relative to vehicle-treated tumors (Fig 7C).

Discussion
Prostate cancer is one of the leading causes of death among men in the United States [19]. Although aggressive efforts toward early detection and treatment decrease mortality rate for prostate cancer, prostate cancer is the second most common cause of cancer death among men yet. Although early-stage prostate cancer requires androgen for growth and thus responds to androgen deprivation therapy, the disease may progress to androgen-independence and may be unresponsive to androgen ablation [20]. Docetaxel-based chemotherapies have shown palliative and survival benefits for patients with castration-resistant prostate cancer, but treatment results are generally unsatisfactory with a median survival time of only 16 to 18 months [21,22]. So it is not only important to develop effective ways of preventing or slowing the formation of castration-resistant prostate cancer, but also to develop new chemotherapeutic agents for the treatment of castration-resistant prostate cancer.
Since ATO was shown to have dramatic effects in patients with acute promyelocytic leukemia [1,2], this agent has also been tested in patients with solid tumors, including prostate cancer [23]. Both ATO and KML001 are trivalent arsenicals and identical substances in solution, with similar cytotoxicity against androgen-independent prostate cancer cells [13]. However, the oral bioavailability and water solubility of KML001 suggest its clinical applicability, compared with ATO. Moreover, KML001 was shown to have higher LD 50 (41.6 mg/kg) in rats, compared with ATO (14.6 mg/kg) [13]. So we tested the ability of KML001 to inhibit the growth and induce cell death of human prostate cancer cell. We found that KML001 treatment reduced the proliferation of all 3 prostate cancer cell lines, being more toxic to androgendependent LNCaP cells than to androgen-independent cells. More importantly, KML001 had an antiproliferative effect on androgen-independent DU145 cells in vitro and in vivo.
ATO acts on malignant cells through a variety of mechanisms, targeting multiple signal transduction pathways and resulting in induction of apoptosis, antiproliferative activity, and antiangiogenesis [3]. Several recent studies have reported that ATO and KML001 may target a telomere/telomerase complex [8][9][10]13]. KML001 binds to telomeric sequences and erodes telomere, resulting in telomere-associated DNA damage induction and telomere attrition. In Cell Death by KML001 in Prostate Cancer Cells addition, ATO was found to induce type II programmed cell death, autophagy, in malignant glioma cells [11,24]. We have shown here that KML001 treatment resulted in the formation of autophagic vacuoles, as documented by electronic microscopy. Moreover, KML001 induced a time-and dose-dependent increase in LC3-II. Using the autophagy inhibitor 3-MA, we corroborated that the mechanism of KML001-induced cell death involves autophagy. Autophagy is not only responsible for cell killing by itself, but also participates in a lethal signaling event inducing apoptosis or necrosis [25,26].
Additionally, we found that the activation of autophagy and apoptosis by KML001 was mediated by oxidative stress. ROS play a pivotal role in mediating the cytotoxicity induced by KML001. Several studies have found that ROS play important roles in regulating both normal cellular processes and disease progression [27][28][29]. In addition, accumulated ROS have been known as the key intermediate for the cytotoxicity induced by chemotherapeutic agents, including ATO [3].
One concern regarding the use of arsenic for clinical applications is its toxicity in humans. Clinical studies have shown that ATO at concentrations less than 2 μM does not induce severe side effects [30,31]. As mentioned above, KML001 was less toxic to rats at the same concentration of ATO [13]. In our study, KML001 had no adverse effect on body weight of DU145 xenograft mice. Our results therefore suggest that KML001 may be clinically useful in patients with castration-resistant prostate cancer.
Whereas most chemotherapeutic agents are aimed at inhibiting the growth of castrationresistant prostate cancer, androgen-dependent and-independent prostate cancer cells have been reported to be equally susceptible to ATO-induced apoptosis [23]. Moreover, inhibition by ATO was more pronounced in prostate cancer cells expressing androgen receptor than in prostate cancer cells depleted of androgen receptor, and inhibition of androgen receptor activity by ATO and by the androgen receptor antagonist, bicalutamide, was additive [24]. These results may warrant the future assessment of the effects of KML001, alone or in combination with androgen deprivation therapy, on the progression of androgen-dependent LNCaP to androgen independence in a nude mice xenograft model.

Conclusions
We have shown here that exposure of androgen-dependent and-independent prostate cancer cells to KML001 activated both apoptosis and autophagic cell death through oxidative stress. In addition, KML001 had an antiproliferative effect on androgen-independent DU145 cells in vivo.