*Article* **Anti-Cancer Activity of As4O<sup>6</sup> and its E**ffi**cacy in a Series of Patient-Derived Xenografts for Human Cervical Cancer**

**Joseph J. Noh 1,**† **, Myeong-Seon Kim 2,**† **, Young-Jae Cho 3,**† **, Soo-Young Jeong <sup>1</sup> , Yoo-Young Lee <sup>1</sup> , Ji-Yoon Ryu <sup>3</sup> , Jung-Joo Choi <sup>3</sup> , Illju Bae <sup>4</sup> , Zhaoyan Wu <sup>4</sup> , Byoung-Gie Kim 1,\*, Jae Ryoung Hwang 3,\* and Jeong-Won Lee 1,\***


Received: 23 September 2020; Accepted: 16 October 2020; Published: 19 October 2020

**Abstract:** Purpose: To investigate the anti-cancer effects of tetraarsenic hexoxide (TAO, As4O6) in cervical cancer cell lines and in a series of patient-derived xenograft (PDX) mouse models. Methods: Human cervical cancer cell lines, including HeLa, SiHa and CaSki, and human umbilical vein endothelial cells (HUVECs), were used to evaluate the anti-cancer activity of TAO. Cellular proliferation, apoptosis, and enzyme-linked immunosorbent assay (ELISA) for matrix metallopeptidase 2 (MMP-2) and 9 (MMP-9) were assessed. The tumor weights of the PDXs that were given TAO were measured. The PDXs included primary squamous cell carcinoma, primary adenocarcinoma, recurrent squamous cell carcinoma, and recurrent adenocarcinoma. Results: TAO significantly decreased cellular proliferation and increased apoptosis in cervical cancer cell lines and HUVEC. The functional studies on the cytotoxicity of TAO revealed that it inhibited the activation of Akt and vascular endothelial growth factor receptor 2 (VEGFR2). It also decreased the concentrations of MMP-2 in both cervical cancer cell lines and HUVECs. Active caspase-3 and p62 were both increased by the treatment of TAO, indicating increased rates of apoptosis and decreased rates of autophagy, respectively. In vivo studies with PDXs revealed that TAO significantly decreased tumor weight for both primary squamous cell carcinoma and adenocarcinoma of the cervix. However, this anti-cancer effect was not seen in PDXs with recurrent cancers. Nevertheless, the combination of TAO with cisplatin significantly decreased tumor weight in PDX models for both primary and recurrent cancers. Conclusions: TAO exerted inhibitory effects on angiogenesis, cellular migration, and autophagy, and it showed stimulatory effects on apoptosis. Overall, it demonstrated anti-cancer effects in animal models for human cervical cancer.

**Keywords:** cervical cancer; tetraarsenic hexoxide; patient-derived xenograft; autophagy; cisplatin

#### **1. Introduction**

Cervical cancer is one of the most common cancers among women worldwide, with almost half a million new cases occurring in each year. In 2015, 526,000 women were diagnosed with cervical cancer and the estimated number of deaths caused by the disease was 239,000 [1]. Cervical cancer patients are prone to developing pelvic recurrence or distant metastasis. A 10–20% recurrence rate has been reported following primary surgery or radiotherapy in women with stage IB–IIA cervical cancer with no evidence of lymph node involvement, while up to 70% of patients with nodal metastases were reported to relapse [2,3]. Because of the unfavorable prognosis of the disease and its high recurrence rate, cervical cancer continues to be a major public health problem, despite widespread screening methods [4]. Various treatment modalities including chemotherapy and radiotherapy have been developed, but none have demonstrated promising results thus far. In order to develop a new approach to improve the prognosis of cervical cancer, researchers have started to investigate various non-chemotherapeutic agents, such as arsenic trioxide (As2O3) and tetraarsenic hexoxide (As4O6, TAO).

Arsenic is a naturally occurring substance that has been used as a medicinal agent for more than 2400 years to treat a variety of medical conditions ranging from infectious disease to cancer [5]. It is stable in dry air, but the surface oxidizes slowly in moist air to give a bronze tarnish and, finally, a black covering to the element. When heated in the air, it ignites to form arsenic trioxide and tetraarsenic hexoxide. In traditional Chinese medicine, arsenic trioxide is recorded in the Compendium of Materia Medica as having therapeutic benefits. Because of the toxic side effects and the introduction of modern radiotherapy and chemotherapy, Western medicine has abandoned the use of arsenic as a treatment for cancer. However, its therapeutic effect on leukemia has initiated a re-awakening of interest in arsenic compounds. Studies have demonstrated that TAO induces apoptosis in hematopoietic and non-hematopoietic tumor cells, eventually gaining approval from the U.S. Food and Drug Administration (FDA) to treat acute promyelocytic leukemia (APL) [6]. The molecular formula of the drug substance in the solid state is As2O<sup>3</sup> (molecular weight of 197.84 g mol−<sup>1</sup> ). Because arsenic trioxide is poorly soluble in pure water, inactive ingredients, such as sodium hydroxide, are added to increase its solubility. Under normal conditions (room temperature and atmospheric pressure), solid arsenic trioxide is present in the form of As4O<sup>6</sup> (dimeric As2O3) and only dissociates into monomeric As2O<sup>3</sup> above 800 ◦C. Upon dissolution of arsenic trioxide in aqueous media, both As2O<sup>3</sup> and As4O<sup>6</sup> are converted into the same arsenic species. It is hypothesized that its anti-cancer effects are mediated by the induction of cellular differentiation, tumor cell apoptosis, degradation of specific transcripts, inhibition of tumor cell growth, modulation of redox balance, and abrogation of vascular networks that cause blood flow to shut down, subsequently causing cell necrosis [7–10]. In an effort to add further evidence to the body of literature suggesting the anti-cancer effects of TAO, the present study was designed to demonstrate the effectiveness of TAO in a series of patient-derived xenografts (PDXs) for cervical cancer, including primary and recurrent patients.

#### **2. Materials and Methods**

#### *2.1. Cell Lines and Tetraarsenic Hexoxide*

Three different cervical cancer cell lines, SiHa (*Homo sapiens* uterine cervix, squamous cell carcinoma: HTB-35), HeLa (*Homo sapiens* uterine cervix, adenocarcinoma: CCL-2), CaSki (*Homo sapiens* uterine cervix, derived from metastatic site of small intestine, epidermoid carcinoma: CRL-1550), and human umbilical vein endothelial cells (HUVECs, CRL-1730) were obtained (American Type Culture Collection, Manassas, VA, USA). Cells were maintained in Dulbecco's modified eagle medium (DMEM), minimal essential medium (MEM), and Roswell Park Memorial Institute (RPMI) medium containing 10% fetal bovine serum (FBS) with 100 units/mL penicillin and 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA) for HeLa, SiHa, and CaSki, respectively. They were grown at 37 ◦C in a 5% CO<sup>2</sup> incubator. HUVECs were grown in an endothelial cell growth medium 2 (EGM-2) bullet kit (Lonza, Basel, Switzerland). TAO was obtained from CHEMAS (Seoul, South Korea). A 1%

concentration of TAO solution in distilled water was made by heating for 4 h at 90–100 ◦C and was then filtered through a 0.2 µm filter.
