**2. Novel arsenic-based combinatorial anticancer therapy**

Based on the promising effect of arsenic on apoptosis in cancer cells, it has been further utilized as a combinatorial drug with other chemotherapeutic agents and/or molecular-targeted drugs to gain its anticancer effect in various types of cancer. The combination drugs, possible target molecules, molecular basis underlying combination treatment-induced apoptosis, and combination indices (CI) for each cancer are summarized in **Figure 1** and **Table 1**.

#### **2.1. Solid cancer**

#### *2.1.1. Colon cancer*

Lee et al. reported that sulindac, a nonsteroidal anti-inflammatory drug (NSAID), enhances ATO-induced apoptosis by inhibiting NF-κB activation mediated through the blocking of phosphorylation and degradation of IκB-alpha in HCT-116 cells [6]. In addition, Cai et al. reported that combined ATO-PI3K inhibitor LY294002 treatment synergistically suppresses the proliferation of colon cancer cell lines, where ATO decreases Hh pathway transcription factor Gli1 and its downstream gene expression including *BCL2* and *CCND1* [7].

**Figure 1.** Molecular mechanism by which ATO exerts an anticancer effect synergistically with other therapeutics. (A) In most cancer cells, ATO generates intracellular ROS, which potentially triggers activation of the apoptotic signaling pathway. A glutathione synthesis inhibitor BSO enhances the effect of ATO-induced ROS generation by depleting GSH [43, 60], while a platinum drug CDDP cooperatively enhances ROS generation [12]. ROS-induced ER stress as well as MAPK phosphorylation can occur in mitochondrial dysfunction, which subsequently activates caspase-3/caspase-7 and induces apoptosis. (B) Arsenite methyltransferase (As3MT)-mediated metabolic methylation of ATO decreases methyl donors (*S*-adenosylmethionine (SAM)) and increases its metabolite *S*-adenosyl-l-homocysteine (SAH), which may inhibit DNA methyltransferase activity of DNMT. ATO itself downregulates gene expression of DNMTs [117]. Consequently, promoter demethylation of silenced genes, including *miR-155*, *miR-200c*, secreted frizzled-related protein-1 (*SFRP1*), and *ERα*, upregulates their gene expression, which may exert anticancer activities [22, 118–120]. The demethylation effect of ATO and other anticancer therapeutics may cooperatively induce apoptosis in cancer cells. (C) In APL cells, ATO binds to PML, while ATRA binds to RARα. Thus, combined ATO-ATRA treatment synergistically induces proteasomal degradation of PML-RARα oncoprotein, differentiation, and subsequent apoptosis [73]. ATO also can induce proteasomal degradation of oncoproteins including chimeric protein BCR-ABL (generated in CML cells), Tax (HTLV-I-encoded protein), and NPM1 (frequently mutated in AML cells). ATO and combination agents are indicated in

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the Double Square. MMA, monomethylarsonous acid; DMA, dimethylarsinous acid.

#### *2.1.2. Prostate cancer*

Therapeutics in prostate cancer is based on the progression stage of the cancer, and radiation therapy is widely utilized for treatment. ATO was reported to enhance the radiation sensitivity of androgen-dependent (LNCaP) and androgen-independent (PC-3) human prostate cancer cells by mediating inhibition of the Akt/mTOR signaling pathway both in vitro and in vivo [8]. As shown in colon cancer, it has been reported that ATO inhibits the proliferation of the prostate cancer cell line PC-3 by suppressing the Hh signaling pathway and the tumor suppression effect was further enhanced by a classic Hh pathway inhibitor cyclopamine in vivo [9]. Furthermore, Arsenic-Based Anticancer-Combined Therapy: Novel Mechanism Inducing Apoptosis of Cancer… http://dx.doi.org/10.5772/intechopen.74824 35

of ATO have revealed the molecular mechanism by which ATO exerts an anticancer effect in both solid cancer and hematological malignancies (see below section). Thus, accumulating evidence implicates ATO and/or other arsenicals in clinical use as a promising drug to treat cancer patients. Besides ATO monotherapy, there is a growing body of evidence that ATO may be a favorable drug when combined with not only conventional anticancer therapeutics including radiation and chemotherapy but also recently developed molecular-targeted drugs. In one example, the combined treatment of all-*trans*-retinoic acid (ATRA) with ATO has been shown to synergistically induce apoptosis of APL cells and clinically shown to be better outcome and less toxicity than the combined treatment of ATRA with chemotherapy in the treatment of patients with APL [4, 5]. This chapter summarizes the anticancer effect of ATO-based combination therapies in different types of solid cancer and hematological malignancies. Furthermore, the molecular mechanism by which ATO-based combination therapies

Based on the promising effect of arsenic on apoptosis in cancer cells, it has been further utilized as a combinatorial drug with other chemotherapeutic agents and/or molecular-targeted drugs to gain its anticancer effect in various types of cancer. The combination drugs, possible target molecules, molecular basis underlying combination treatment-induced apoptosis, and combi-

Lee et al. reported that sulindac, a nonsteroidal anti-inflammatory drug (NSAID), enhances ATO-induced apoptosis by inhibiting NF-κB activation mediated through the blocking of phosphorylation and degradation of IκB-alpha in HCT-116 cells [6]. In addition, Cai et al. reported that combined ATO-PI3K inhibitor LY294002 treatment synergistically suppresses the proliferation of colon cancer cell lines, where ATO decreases Hh pathway transcription

Therapeutics in prostate cancer is based on the progression stage of the cancer, and radiation therapy is widely utilized for treatment. ATO was reported to enhance the radiation sensitivity of androgen-dependent (LNCaP) and androgen-independent (PC-3) human prostate cancer cells by mediating inhibition of the Akt/mTOR signaling pathway both in vitro and in vivo [8]. As shown in colon cancer, it has been reported that ATO inhibits the proliferation of the prostate cancer cell line PC-3 by suppressing the Hh signaling pathway and the tumor suppression effect was further enhanced by a classic Hh pathway inhibitor cyclopamine in vivo [9]. Furthermore,

exert a proapoptotic effect in cancer cells is discussed.

34 Current Understanding of Apoptosis - Programmed Cell Death

**2.1. Solid cancer**

*2.1.1. Colon cancer*

*2.1.2. Prostate cancer*

**2. Novel arsenic-based combinatorial anticancer therapy**

nation indices (CI) for each cancer are summarized in **Figure 1** and **Table 1**.

factor Gli1 and its downstream gene expression including *BCL2* and *CCND1* [7].

**Figure 1.** Molecular mechanism by which ATO exerts an anticancer effect synergistically with other therapeutics. (A) In most cancer cells, ATO generates intracellular ROS, which potentially triggers activation of the apoptotic signaling pathway. A glutathione synthesis inhibitor BSO enhances the effect of ATO-induced ROS generation by depleting GSH [43, 60], while a platinum drug CDDP cooperatively enhances ROS generation [12]. ROS-induced ER stress as well as MAPK phosphorylation can occur in mitochondrial dysfunction, which subsequently activates caspase-3/caspase-7 and induces apoptosis. (B) Arsenite methyltransferase (As3MT)-mediated metabolic methylation of ATO decreases methyl donors (*S*-adenosylmethionine (SAM)) and increases its metabolite *S*-adenosyl-l-homocysteine (SAH), which may inhibit DNA methyltransferase activity of DNMT. ATO itself downregulates gene expression of DNMTs [117]. Consequently, promoter demethylation of silenced genes, including *miR-155*, *miR-200c*, secreted frizzled-related protein-1 (*SFRP1*), and *ERα*, upregulates their gene expression, which may exert anticancer activities [22, 118–120]. The demethylation effect of ATO and other anticancer therapeutics may cooperatively induce apoptosis in cancer cells. (C) In APL cells, ATO binds to PML, while ATRA binds to RARα. Thus, combined ATO-ATRA treatment synergistically induces proteasomal degradation of PML-RARα oncoprotein, differentiation, and subsequent apoptosis [73]. ATO also can induce proteasomal degradation of oncoproteins including chimeric protein BCR-ABL (generated in CML cells), Tax (HTLV-I-encoded protein), and NPM1 (frequently mutated in AML cells). ATO and combination agents are indicated in the Double Square. MMA, monomethylarsonous acid; DMA, dimethylarsinous acid.


Tai et al. reported that combined ATO-mTOR inhibitor RAD001 (everolimus) treatment synergistically induces both apoptosis and autophagy in prostate cancer cells, where enhanced autophagic cell death was accompanied by increased Beclin1 mRNA stability as well as upregulation of ATG5-ATG12 conjugate, Beclin1, and LC3-LC2 [10]. Importantly, the study showed that ATO-RAD001 combinatorial treatment more significantly suppresses LNCaP xenograft

ATL — Degradation of HTLV-I

**Combination regime Cancer type CI value Mechanism of action Ref.**

AML — ROS-mediated

ATO + bortezomib (BOR) Multiple myeloma 0.4–0.64 Synergistic effect of ATO/BOR

Lymphoma and leukemia

Chronic myelogenous leukemia (CML)

Primary effusion lymphoma

**Table 1.** Summary of representative ATO-based combination strategies to treat cancer.

Combination index value (CI); synergism is indicated by CI < 1.

Ovarian cancer — GSH depletion, increased

Mantle cell lymphoma <1.0 Inhibition of NF-κB activity,

Lung cancer — GSH depletion [43] Glioma — GSH depletion [60]

Arsenic-Based Anticancer-Combined Therapy: Novel Mechanism Inducing Apoptosis of Cancer…

intracellular ROS generation, and activation of oxidative stress-related pathway

phosphorylation of JNK and BIMEL and induction of intrinsic apoptosis

with p38 inhibitor (SB203580) on Bcl-2 downregulation and apoptosis in MM cell lines

decreases in cyclin D1 and Bcl-2 expression, and decreased interaction of Mcl-1

transactivator protein (Tax)

— Inhibition of NF-κB activity [111]

with Bak

— Suppressive activity of CML leukemia-initiating cells

— ROS-mediated phosphorylation of JNK and upregulation of

death receptor 5

[17]

37

http://dx.doi.org/10.5772/intechopen.74824

[87]

[88]

[97]

[114, 115]

[108]

[109, 110]

ATO + buthionine sulfoximine (BSO)

ATO + interferon-α

(IFN-α)

OSCC is the most common head and neck neoplasm and is highly associated with poor prognosis, despite advances that have been made in diagnostic and therapeutic strategies such as surgery, chemotherapy, and radiotherapy. ATO was reported as a combinatorial drug with radiotherapy [11] and a platinum-based antineoplastic drug cisplatin (CDDP) [12], both of which

tumor proliferation than monotherapy without enhancing weight loss [10].

*2.1.3. Oral cancer (oral squamous cell carcinoma (OSCC))*

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**Table 1.** Summary of representative ATO-based combination strategies to treat cancer.

Tai et al. reported that combined ATO-mTOR inhibitor RAD001 (everolimus) treatment synergistically induces both apoptosis and autophagy in prostate cancer cells, where enhanced autophagic cell death was accompanied by increased Beclin1 mRNA stability as well as upregulation of ATG5-ATG12 conjugate, Beclin1, and LC3-LC2 [10]. Importantly, the study showed that ATO-RAD001 combinatorial treatment more significantly suppresses LNCaP xenograft tumor proliferation than monotherapy without enhancing weight loss [10].

#### *2.1.3. Oral cancer (oral squamous cell carcinoma (OSCC))*

**Combination regime Cancer type CI value Mechanism of action Ref.**

Oral cancer — Inhibition of tumor growth,

Cervical cancer — Suppression of radiation-

Glioma — Increased mitotic arrest

Ovarian cancer 0.63–0.93 Upregulation of *BAX* and *TP53*

Lung cancer 0.5–0.6 Increases in Bax and decreases

Glioma — Suppression of cancer stem cell

AML/NPM1-mutated — Degradation of nucleophosmin

Hepatoma — Reduced GSH level [67]

signaling pathway

translocation

pathways

caspase-3

by only ATO)

caspase-3

(CSC) properties

— ATO and APL differentially induce proteasomal degradation of PML-RARα

— Co-inhibition of FLT3 signaling pathways

(NPM1)

— Degradation of HTLV-I

transactivator protein (Tax)

in Bcl-2 and clusterin

Breast cancer — Bcl-2/Bax ratio [23]

angiogenesis, and metastasis

induced MMP-9 expression, ROS generation-induced MAPKs activation, and Bax

and regulation of PI3K/Akt and ERK1/ERK2 signaling

in Bcl-2 protein level, and constitutive activation of

and downregulation of *HIF1A*, *IGF1R*, *MET*, and *AR* (effects

[8]

[11]

[18, 19]

[53]

[12]

[15]

[44]

[21]

[61]

[73]

[85]

[86]

[118]

ATO + radiotherapy Prostate cancer — Inhibition of Akt/mTOR

36 Current Understanding of Apoptosis - Programmed Cell Death

ATO + cisplatin (CDDP) Oral cancer 0.34–0.92 ROS generation, decrease

) + CDDP Cervical cancer — Synergistic activation of

Lung adenocarcinoma — Breast cancer —

Acute promyelocytic leukemia (APL)

Adult T-cell leukemia (ATL)/RARα-positive

Acute myeloid leukemia (AML)/ FLT3-ITD

TAO (As<sup>4</sup> O6

acid (ATRA)

ATO + all-trans-retinoic

OSCC is the most common head and neck neoplasm and is highly associated with poor prognosis, despite advances that have been made in diagnostic and therapeutic strategies such as surgery, chemotherapy, and radiotherapy. ATO was reported as a combinatorial drug with radiotherapy [11] and a platinum-based antineoplastic drug cisplatin (CDDP) [12], both of which are the most standard therapies for OSCC. Since ATO/CDDP-induced apoptosis was almost completely abrogated by NAC, ROS generation may be closely associated with the tumor suppression effect (**Figure 1A**) [12]. Recent publications also implicated the therapeutic application of arsenic in the treatment for OSCC. Wang et al. reported that nicotinamide phosphoribosyltransferase (NAMPT) increases in patients with OSCC and a NAMPT inhibitor FK866 and ATO cooperatively induced apoptosis and depletes intracellular nicotinamide adenine dinucleotide levels in OSCC cell lines [13]. Tsai et al. showed that the combined ATO-dithiothreitol (DTT) treatment increases proapoptotic molecules Bax and Bak and decreases Bcl-2 and p53, which leads to a significant cell death of oral cancer cells but not the non-tumor cells [14].

better prognosis (**Figure 1B**) [22]. Subsequently, combined ATO and antiestrogen tamoxifen (TAM) therapy coordinately suppressed tumor growth of a human breast cancer cell line MDA-MB-435S both in vitro and in vivo [22]. ATO was reported to enhance 89Sr radiation treatment-induced apoptosis by partly modulating the Bcl-2/Bax ratio [23]. Guilbert et al. reported that ATO suppresses rapamycin (specific mTOR inhibitor)-induced phosphorylation of both ERK and Akt (Ser473), which leads to enhancement of the anticancer effect of rapamycin in vivo [24]. Cotylenin A (CN-A), a plant growth regulator, was reported to exert a favorable antitumor effect on breast cancer cells when it was co-incubated with ATO in vitro [25]. The combined CN-A-ATO treatment decreased survivin expression and increased caspase-7 expression by partly mediating ROS generation [25]. It has been reported that melatonin, a known natural antioxidant, enhances ATO-induced apoptosis by mediating ROS generation-induced MAPK activation including p38 and JNK in human breast cancer cell lines MDA-MB-231 and SK-BR-3 [26]. They also showed that mTOR inhibitor rapamycin further enhances the ATO-melatonin-induced apoptosis [26]. In addition to inorganic arsenite ATO, its intermediate metabolites monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) exert more cytotoxicity toward breast cancer cells than ATO, implicating application of the arsenite-related intermediates in anticancer therapy for breast cancer [27]. The combined intermediates MMAIII and DMAIII cryptotanshinone (a natural quinoid diterpene isolated from *Salvia miltiorrhiza* roots) strongly induce apoptosis by mediating endoplasmic

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Hepatocellular carcinoma (HCC) is the most common primary liver malignancy, which is the sixth most common type of cancer worldwide. Sorafenib, a known multikinase inhibitor, can extend the survival rate of patients with advanced HCC. ATO was reported to synergize with sorafenib to inhibit the proliferation and promote the apoptosis of HCC cells by diminishing the sorafenib-induced activation of Akt and/or its downstream factors, including glycogen synthase kinase-3β, mTOR, ribosomal protein S6 kinase, and eukaryotic translation initiation factor 4E-binding protein 1 [28]. ATO was also reported to potentiate the anticancer effect of genistein [29], 3′-azido-3′-deoxythymidine (AZT) [30], oridonin [31], MDM2 inhibitor nutlin-3 [32], metformin [33–34], survivin mutant (T34A) [35], shikonin [36], and andrographolide [37] in HCC cells.

Lung cancer is the most common type of cancer worldwide. As indicated in colon cancer, there are several reports regarding the synergistic induction of apoptosis by an NSAID sulindac and ATO in human lung cancer cell lines [38, 39]. Combined ATO-sulindac treatment induced apoptosis of human non-small cell lung cancer (NSCLC) cell line A549 by mediating the mitochondrial pathway and the NF-κB pathway [38] and by mediating p53-induced downregulation of survivin [39]. It has also been reported that combined ATO-sulindac treatment induces synergistic augmentation of cytotoxicity in both human NSCLC cell lines NCI-H157 and NCI-H1299

dent Bcl-xL phosphorylation, respectively [40, 41]. Indomethacin, a nonselective cyclooxygenase inhibitor (a structural isoform of sulindac), was also shown to enhance the ATO-induced cytotoxic effect in A549 cells by mediating activation of ERK and/or p38 MAPKs [42]. Han et al.


reticulum (ER) stress and/or ROS generation in MCF-7 cells [27].

by mediating ROS-induced MAPK phosphorylation and via c-Jun NH<sup>2</sup>

*2.1.7. HCC and bile duct carcinoma (cholangiocarcinoma)*

*2.1.8. Lung cancer*

#### *2.1.4. Ovarian cancer*

The rate of the mortality from ovarian cancer is highest among malignant tumors of the female genital organs. As indicated in other types of cancer, ATO was reported to exert synergistic cytotoxic effects against ovarian cancer cells when it was combined with CDDP, one of the standard chemotherapeutics for ovarian cancer, and/or mTOR inhibitor RAD001 [15, 16]. Ong et al. reported that both buthionine sulfoximine and ascorbic acid differentially enhance ATO-mediated cell killing by mediating GSH depletion and the oxidative stress-related pathway, respectively [17].
