*2.2.1. Acute promyelocytic leukemia (APL)*

Patients with newly diagnosed APL, also known as a AML M3 subtype with APL-specific chromosomal translocation t(15;17) (q22;q21) and PML/RARα chimeric fusion protein, are mostly cured after standard ATRA with chemotherapy, while toxicity and refractoriness to the treatment are observed in some patients. Accumulating evidence shows the superiority of novel combined ATRA and ATO therapy for the treatment of patients with APL in terms of event-free survival, relapse-free survival, and less hematologic toxicity, compared to ATRA with chemotherapy [4, 5, 69–72]. The molecular basis underlying synergistic effects between ATRA and ATO has been biologically demonstrated. ATRA and ATO differentially bind PML/RARα protein, the proteasomal degradation of which readily induces terminal differentiation, and subsequent apoptosis in APL cells (**Figure 1C**) [73]. In addition to the effect of ATO on the PML moiety, ATO-induced anticancer activities including inhibition of leukemic progenitor self-replication and antiangiogenic effects might be involved in the antileukemic activity. The combined ATRA with intravenous administration of ATO has been developed to combine ATRA with oral As4S4 administration as a routine treatment option for appropriate patients [74]. Moreover, Wang et al. showed that the combination of low concentrations of As4S4 and As3+ enhanced degradation of the PML/RARα oncoprotein and subsequent apoptosis [75]. Other modified combination regimes have been demonstrated using in vitro experimental models [76–82]. Jung et al. reported that the Src family kinase inhibitor PP2 enhances differentiation of APL cells induced by ATRA-ATO treatment [76]. Rogers et al. reported that vitamin D3 potentiates the antitumorigenic effects of ATO in HL-60 cells (PML/RARα-negative APL cell line) by enhancing nuclear DNA fragmentation [77]. The antileukemic activity of ATO was also enhanced by the combination strategies with granulocytemonocyte colony stimulation factor [78], a noncalcemic vitamin D analog 19-Nor-1,25(OH)2D2 [79], *N*-(beta-Elemene-13-yl) tryptophan methyl [80], a selective inhibitor of epidermal growth factor receptor (EGFR) gefitinib [81], and high-dose vitamin C (ascorbic acid) [82], all of which enhance ATO-induced differentiation and apoptosis of APL cells.

*2.2.3. Multiple myeloma (MM)*

*2.2.4. Chronic myelogenous leukemia (CML)*

*2.2.5. Other leukemia and lymphoma*

ATO, melphalan, and ascorbic acid (AA) combination therapy (MAC) is a therapeutic option for patients with relapsed or refractory MM [94]. Grad et al. initially showed that clinically relevant doses of AA decrease GSH levels and potentiate ATO-mediated cell death of MM cell lines [95]. Current therapeutics for MM, such as a proteasome inhibitor, namely, bortezomib (BOR) and carfilzomib, and immunomodulatory drugs, namely, thalidomide, lenalidomide (LEN), and pomalidomide, have successfully improved the patient survival, though MM remains an incurable disease [96]. In view of the current MM therapeutics, ATO was reported to enhance the anti-myeloma cytotoxicity of BOR [97] and sensitivity of MM cells to lenalidomide (LEN) [98]. Wen et al. showed that the enhanced cytotoxicity of ATO-BOR is associated with augmented STAT3 inhibition, JNK activation, and upregulation of Bim, p21, p27, and p53 as well as downregulation of Bcl-2 [97]. Jian et al. showed that ATO upregulates cereblon, the antimyeloma target of LEN, thus potentiating the sensitivity of MM cells [98]. The anti-myeloma activity of ATO was also enhanced by the combination strategies with a vitamin E analog Trolox [99], a specific MEK inhibitor PD325901 [100], a natural quinoid diterpene cryptotanshinone (also known as STAT3 inhibitor) [101, 102], and a phytochemical sulforaphane [103].

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Recent therapeutics for CML were developed by targeting the Bcr-Abl fusion protein generated from a Philadelphia (Ph) chromosome with reciprocal translocation of chromosomes 9 and 22. Despite the advances in CML therapeutics including Bcr-Abl tyrosine kinase inhibitors (TKIs), TKI therapy can produce a subpopulation of CML cells with a Bcr-Abl gene mutation that leads to resistance to TKI therapy, which results in a poorer prognosis in 10–15% of patients with CML. Several reports indicated the efficacy of ATO-based combined therapy for CML cells [104–108]. Du et al. reported that combined treatment of ATO with imatinib, which is the first approved TKI, coordinately enhances apoptosis of CML cells by mediating intrinsic (upregulation of *BAX*), extrinsic (upregulation of *TNFR1*, *CASP8*, and *CASP10*), and ER stressrelated pathways (*HSPA5* and *DDIT3*) [104]. Wang et al. also showed the additive effect of ATOnilotinib, a second-line TKI agent, on the proliferation and differentiation of primary leukemic cells from patients with CML in blast crisis [105]. In addition, combined nilotinib-ATO treatment induces ER stress-mediated apoptosis in imatinib-resistant K562 cells by mediating JNK activation [106]. Li et al. reported that anti-miR-21 oligonucleotide sensitizes K562 cells to ATO and enhances ATO-induced apoptosis probably by mediating upregulation of programmed cell death 4 (PDCD4) [107]. In a CML mice model, combined treatment with ATO and interferon-α (IFN-α) was reported to be superior to imatinib [108] in terms of overall survival of secondary recipients, indicating that ATO-IFN-α may exhaust the activity of CML leukemia-initiating cells.

As the efficacy was shown in a CML mice model [108], ATO and IFN-α synergized to induce cell cycle arrest and apoptosis in adult T-cell leukemia/lymphoma (ATL)-derived human T-cell lymphotropic virus type I (HTLV-I)-transformed cells [109, 110]. El-Sabban et al. reported that combined ATO-IFN-α treatment induces the degradation of Tax, which is the

#### *2.2.2. Acute myeloid leukemia (AML)*

AML is a malignant disease of the bone marrow, where juvenile leukocytes are arrested in an early stage of differentiation. It has been reported that AML patients with FLT mutations including FLT3-internal tandem duplication (FLT3-ITD) had significantly shorter overall and disease-free survival [83]. Takahashi et al. reported that combined treatment with ATO and FLT3-specific inhibitor AG1296 synergistically induces apoptosis in FLT3-ITD-positive cells, but not in Flt3 wild-type cells [84]. The combined ATO with ATRA, a novel standard treatment for patients with APL, was shown to exert synergistic cytotoxicity against FLT3-ITD AML cells via co-inhibition of FLT3 signaling pathways [85]. In addition, ATO-ATRA was shown to induce apoptosis of NPM1-mutated AML cells by targeting nucleophosmin (NPM1) oncoprotein, whose mutation possibly represents a therapeutic target because of high frequency in >30% AML [86]. As indicated in lung cancer and glioma, BSO was shown to enhance the ATOinduced anticancer effect by mediating ROS generation in AML cells [87] and other leukemic/lymphoma cells [88], suggesting that combined ATO-BSO treatment would be one of the attractive alternative therapies for cancer treatment. It has also been reported that combined treatment with ATO and dichloroacetate [89], azacytidine [90], rapamycin [91], and aclacinomycin A [92] enhances apoptosis in AML cells. Wang et al. reported that ethacrynic acid and a derivative enhance apoptosis in ATO-treated myeloid leukemia and lymphoma cell lines; this combination treatment generates high levels of ROS, activates JNK, and subsequently decreases the protein level of antiapoptotic molecule Mcl-1 [93].

#### *2.2.3. Multiple myeloma (MM)*

cured after standard ATRA with chemotherapy, while toxicity and refractoriness to the treatment are observed in some patients. Accumulating evidence shows the superiority of novel combined ATRA and ATO therapy for the treatment of patients with APL in terms of event-free survival, relapse-free survival, and less hematologic toxicity, compared to ATRA with chemotherapy [4, 5, 69–72]. The molecular basis underlying synergistic effects between ATRA and ATO has been biologically demonstrated. ATRA and ATO differentially bind PML/RARα protein, the proteasomal degradation of which readily induces terminal differentiation, and subsequent apoptosis in APL cells (**Figure 1C**) [73]. In addition to the effect of ATO on the PML moiety, ATO-induced anticancer activities including inhibition of leukemic progenitor self-replication and antiangiogenic effects might be involved in the antileukemic activity. The combined ATRA with intravenous administration of ATO has been developed to combine ATRA with oral As4S4 administration as a routine treatment option for appropriate patients [74]. Moreover, Wang et al. showed that the combination of low concentrations of As4S4 and As3+ enhanced degradation of the PML/RARα oncoprotein and subsequent apoptosis [75]. Other modified combination regimes have been demonstrated using in vitro experimental models [76–82]. Jung et al. reported that the Src family kinase inhibitor PP2 enhances differentiation of APL cells induced by ATRA-ATO treatment [76]. Rogers et al. reported that vitamin D3 potentiates the antitumorigenic effects of ATO in HL-60 cells (PML/RARα-negative APL cell line) by enhancing nuclear DNA fragmentation [77]. The antileukemic activity of ATO was also enhanced by the combination strategies with granulocytemonocyte colony stimulation factor [78], a noncalcemic vitamin D analog 19-Nor-1,25(OH)2D2 [79], *N*-(beta-Elemene-13-yl) tryptophan methyl [80], a selective inhibitor of epidermal growth factor receptor (EGFR) gefitinib [81], and high-dose vitamin C (ascorbic acid) [82], all of which

AML is a malignant disease of the bone marrow, where juvenile leukocytes are arrested in an early stage of differentiation. It has been reported that AML patients with FLT mutations including FLT3-internal tandem duplication (FLT3-ITD) had significantly shorter overall and disease-free survival [83]. Takahashi et al. reported that combined treatment with ATO and FLT3-specific inhibitor AG1296 synergistically induces apoptosis in FLT3-ITD-positive cells, but not in Flt3 wild-type cells [84]. The combined ATO with ATRA, a novel standard treatment for patients with APL, was shown to exert synergistic cytotoxicity against FLT3-ITD AML cells via co-inhibition of FLT3 signaling pathways [85]. In addition, ATO-ATRA was shown to induce apoptosis of NPM1-mutated AML cells by targeting nucleophosmin (NPM1) oncoprotein, whose mutation possibly represents a therapeutic target because of high frequency in >30% AML [86]. As indicated in lung cancer and glioma, BSO was shown to enhance the ATOinduced anticancer effect by mediating ROS generation in AML cells [87] and other leukemic/lymphoma cells [88], suggesting that combined ATO-BSO treatment would be one of the attractive alternative therapies for cancer treatment. It has also been reported that combined treatment with ATO and dichloroacetate [89], azacytidine [90], rapamycin [91], and aclacinomycin A [92] enhances apoptosis in AML cells. Wang et al. reported that ethacrynic acid and a derivative enhance apoptosis in ATO-treated myeloid leukemia and lymphoma cell lines; this combination treatment generates high levels of ROS, activates JNK, and subsequently

enhance ATO-induced differentiation and apoptosis of APL cells.

decreases the protein level of antiapoptotic molecule Mcl-1 [93].

*2.2.2. Acute myeloid leukemia (AML)*

42 Current Understanding of Apoptosis - Programmed Cell Death

ATO, melphalan, and ascorbic acid (AA) combination therapy (MAC) is a therapeutic option for patients with relapsed or refractory MM [94]. Grad et al. initially showed that clinically relevant doses of AA decrease GSH levels and potentiate ATO-mediated cell death of MM cell lines [95]. Current therapeutics for MM, such as a proteasome inhibitor, namely, bortezomib (BOR) and carfilzomib, and immunomodulatory drugs, namely, thalidomide, lenalidomide (LEN), and pomalidomide, have successfully improved the patient survival, though MM remains an incurable disease [96]. In view of the current MM therapeutics, ATO was reported to enhance the anti-myeloma cytotoxicity of BOR [97] and sensitivity of MM cells to lenalidomide (LEN) [98]. Wen et al. showed that the enhanced cytotoxicity of ATO-BOR is associated with augmented STAT3 inhibition, JNK activation, and upregulation of Bim, p21, p27, and p53 as well as downregulation of Bcl-2 [97]. Jian et al. showed that ATO upregulates cereblon, the antimyeloma target of LEN, thus potentiating the sensitivity of MM cells [98]. The anti-myeloma activity of ATO was also enhanced by the combination strategies with a vitamin E analog Trolox [99], a specific MEK inhibitor PD325901 [100], a natural quinoid diterpene cryptotanshinone (also known as STAT3 inhibitor) [101, 102], and a phytochemical sulforaphane [103].

#### *2.2.4. Chronic myelogenous leukemia (CML)*

Recent therapeutics for CML were developed by targeting the Bcr-Abl fusion protein generated from a Philadelphia (Ph) chromosome with reciprocal translocation of chromosomes 9 and 22. Despite the advances in CML therapeutics including Bcr-Abl tyrosine kinase inhibitors (TKIs), TKI therapy can produce a subpopulation of CML cells with a Bcr-Abl gene mutation that leads to resistance to TKI therapy, which results in a poorer prognosis in 10–15% of patients with CML. Several reports indicated the efficacy of ATO-based combined therapy for CML cells [104–108]. Du et al. reported that combined treatment of ATO with imatinib, which is the first approved TKI, coordinately enhances apoptosis of CML cells by mediating intrinsic (upregulation of *BAX*), extrinsic (upregulation of *TNFR1*, *CASP8*, and *CASP10*), and ER stressrelated pathways (*HSPA5* and *DDIT3*) [104]. Wang et al. also showed the additive effect of ATOnilotinib, a second-line TKI agent, on the proliferation and differentiation of primary leukemic cells from patients with CML in blast crisis [105]. In addition, combined nilotinib-ATO treatment induces ER stress-mediated apoptosis in imatinib-resistant K562 cells by mediating JNK activation [106]. Li et al. reported that anti-miR-21 oligonucleotide sensitizes K562 cells to ATO and enhances ATO-induced apoptosis probably by mediating upregulation of programmed cell death 4 (PDCD4) [107]. In a CML mice model, combined treatment with ATO and interferon-α (IFN-α) was reported to be superior to imatinib [108] in terms of overall survival of secondary recipients, indicating that ATO-IFN-α may exhaust the activity of CML leukemia-initiating cells.

#### *2.2.5. Other leukemia and lymphoma*

As the efficacy was shown in a CML mice model [108], ATO and IFN-α synergized to induce cell cycle arrest and apoptosis in adult T-cell leukemia/lymphoma (ATL)-derived human T-cell lymphotropic virus type I (HTLV-I)-transformed cells [109, 110]. El-Sabban et al. reported that combined ATO-IFN-α treatment induces the degradation of Tax, which is the viral transactivator protein that plays a critical role in HTLV-I-induced transformation and apoptosis resistance [110]. Similarly, the enhanced ATO-IFN-α-induced apoptosis was shown in primary effusion lymphoma [111]. Darwiche et al. showed that synergism of ATO-ATRA is especially observed in the HTLV-I-transformed cells expressing RARα protein [112]. In acute lymphoblastic leukemia (ALL), low-dose ATO sensitized glucocorticoid-resistant ALL cells to dexamethasone via an Akt-dependent pathway [113]. Jung et al. and Zhao et al. independently showed the synergistic anticancer effects of ATO with BOR in mantle cell lymphoma, which is an aggressive and highly incurable B-cell non-Hodgkin lymphoma [114, 115]. Ding et al. recently reported that combined treatment of ATO with cucurbitacin B, an effective component of the dichloromethane extraction from *Trichosanthes kirilowii maxim*, synergistically enhances apoptosis by inhibiting STAT3 phosphorylation in Burkitt's lymphoma cell lines both in vitro and in vivo [116].

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