**2.4 Biological studies**

**Table 1** shows the anti-proliferative activity (IC50 values) for **1**, bozepinib and 5-fluorouracil (5-FU) as a reference drug. Compounds were first assayed as antiproliferative agents against the human breast adenocarcinoma cell line MCF-7 (p53 wild type and ras mutated). Compounds **1** and bozepinib were further assayed against the human breast cancer cell line MDA-MB-231 which presents high levels of mutant p53 [28, 30]. The IC50 = 0.166 μM for bozepinib against the human cancerous cell line MDA-MB-231 stands out.

In order to determine the in vitro therapeutic index of the compounds, they were assayed against the non-cancerous human mammary epithelial cell line MCF-1oA. The TI of a drug is defined as the ratio of the toxic dose to the therapeutic dose (in vitro TI = IC50 non-tumor cell line/IC50 tumor cell line) [28]. Bozepinib is more selective against both human breast adenocarcinoma MCF-7 and MDA-MB-231 cancer cell lines (TIs = 5.14 and 11.0, respectively) in relation to the normal one (**Table 2**).

As bozepinib is more active and more selective and more active than its isomer **1**, we decided to carry out the separation of bozepinib into their component enantiomers (resolution). (*S*)-**2** shows higher anti-proliferative effect that of (*R*)-**2** in the MCF-7 cell line. However no differences against the MDA-MB-231 cell line were observed (**Table 3**). The enantioselective cytotoxicity indicates that the


*a All experiments were conducted in duplicate and gave similar results. Data are means ± SEM of three independent determinations. The treatment time was 48 h.*

*b N.D. = not determined.*

*c Taken from Ref. [31].*

#### **Table 1.**

*Anti-proliferative activitiesa for compounds 1 and bozepinib against the cancerous cell lines MCF-7 and MDA-MB-231 and the non-cancerous cell line MCF-10A.*


#### **Table 2.**

*Therapeutic indexes for 1 and bozepinib.*


*a All experiments were conducted in duplicate and gave similar results. The data are means ± SEM of three independent determinations.*

#### **Table 3.**

*Anti-proliferative activities of bozepinib and its enantiomers against the cancerous cell lines MCF-7 and MDA-MB-231.*

enantiomers of some chiral drugs may differ both quantitatively and qualitatively in their biological activity [32, 33]. Moreover, enantiomers can show minimal in vitro but a dramatic in vivo chiral dependency in their antitumor activities [34, 35].

#### *2.4.1 Effect of bozepinib against CSC subpopulations*

The cytotoxic effect of bozepinib was determined on SKBR-3 and MDA-MB 468 breast and HCT-116 colon CSC-enriched subpopulations by growing them into low attachment plates with sphere-forming medium for 72 h. Salinomycin is a selective and potent drug used in the treatment of CSCs, but its use is limited in humans due to considerable toxicity [36]. Thus, we decided to include salinomycin in our studies as reference drug. Bozepinib displayed IC50 values in a similar range to salinomycin in SKBR-3 and MDA-MB 468 cells [30].

CSCs were separated using ALDH activity by FACS. IC50 values were determined in both ALDH positive cells (called ALDH+), ALDH negative cells (called ALDH−) and cells growing in sphere-forming medium without sorter enrichment process [30]. Since there is controversy about the efficacy of sphere-forming cultures to select CSCs [37, 38], we used ALDH activity to enrich cell cultures with clonal ability and stem-like properties. Bozepinib substantially diminished the number and size of spheres at 5 μM and abolished the formation of spheres at 20 μM [30] in subpopulations isolated by ALDH activity. High ALDH activity is associated with metastasis, resistance to chemotherapies and poor prognosis in human cancers, and it has been identified as one of the most specific markers of human CSCs [39].

Apoptosis is also an important factor in the onset of a tumor. It has been reported than CSCs are resistant to apoptosis to ensure succeeding generation [40] and may be on the causes to chemo- or radiotherapy survival. Reactivating death programmes in CSCs may increase efficacy of the treatment. Bozepinib induced a significant level of cell death by apoptosis in the resistant ALDH+ subpopulations from both SKBR-3 and HCT-116 cell lines. Higher levels of apoptosis were detected in the ALDH− subpopulations.

Bozepinib induced the downregulation of important genes involved in Notch and Wnt signaling. These genes contribute to the CSC phenotype when they are deregulated [41]. A downregulation was detected of the co-activator

**135**

morbidity [28].

*Bozepinib: A Promising Selective Derivative Targeting Breast Cancer Stem Cells*

the treatment of determined HER2-related breast cancers [43, 44].

Mastermind-like MAML-2 protein, which decreases Notch signaling, reducing the primary tumor sphere formation and side population in MCF-7 cell line, contributing to the decrease in the number of CSC subpopulations [42]. We also found an evident downregulation of the *NOTCH 3* gene, which has recently been involved in the proliferation of both HER2 positive and negative breast cancer cells, suggesting that targeted suppression of this signaling pathway may be a promising strategy for

The modification of proteins related to the CSC phenotype such as β-catenin, GLI-3, c-MYC and SOX-2 was additionally analysed. This study was carried out in both ALDH+ and ALDH− SKBR-3 breast and HCT-116 colon cancer subpopulations. β-Catenin is a key mediator of the Wnt signaling which has a crucial role in CSCs

The c-MYC oncoprotein was observed in SKBR-3 and HCT-116 cell lines with a high level of expression in ALDH+ subpopulations in comparison with the ALDH− subpopulations. Bozepinib induced a significant decrease of c-MYC level in ALDH+ HCT-116 cells and inhibited its expression in ALDH+ SKBR-3-treated cells. It also inhibited ALDH− subpopulations of both HCT-116 and SKBR-3 cells [30]. In accordance with the high level of stem signaling proteins described for CSCs, higher levels of these proteins in the ALDH+ subpopulations in comparison with ALDHcells were detected. The downregulation of c-MYC activates the inhibition of cancer

The transcription factor SOX2 was detected in HCT-116 ALDH+ subpopulation. However SOX2 was not observed in breast cancer cells, and the expression of SOX2 was very weak in HCT-116 ALDH− subpopulation [30]. Its expression practically disappeared after treatment with bozepinib. SOX2 is involved in the induction and maintenance of pluripotent stem cells and has also been associated with metastases

The acute toxicity profile of bozepinib was determined in BALB/c mice. After 2 weeks, bozepinib was nontoxic to BALB/c mice even at the highest intraperitoneal bolus dose of 200 mg/kg and orally bolus dose of 50 mg/kg. Control mice were treated with vehicle alone. All 50 bozepinib-treated mice remained healthy and gained weight throughout the 15-day observation period, with no evidence of

[45]. β-Catenin expression was detected in ALDH+ SKBR-3 but not in ALDH− SKBR-3 cells. A high level of expression in HCT-116 cell lines with an ALDH+ subpopulations in comparison with the ALDH− in HCT-116 subpopulations was also detected. After bozepinib treatment, β-catenin expression was reduced in ALDH+ HCT-116 subpopulation. Its inhibition in ALDH− HCT-116 cells was also observed. GLI-3, a described target gene transcription repressor of Hedgehog signaling pathway [16], was detected in ALDH− HCT-116 isolated cells and was not present in the ALDH+ cells, denoting that the Hedgehog signaling pathway is involved in the CSC phenotype as previously reported [16]. Bozepinib greatly induced the expression of GLI-3 in both ALDH+ and ALDH− HCT-116 cells. However, no changes at protein level were observed in ALDH+/− subpopulations isolated from the SKBR-3 cell line. Furthermore, the activation of GLI-3 protein corresponded with an inactivation of β-catenin expression in CSC subpopulations after bozepinib treatment in accordance with the previous published studies [46]. Considering that GLI-3 overexpression decreased tumor cell proliferation and induced apoptosis in colon CSCs [47], the GLI-3 induction by bozepinib could be one of the mechanisms by which this drug exerts its antitumor activity in colon CSCs. This hypothesis will

*DOI: http://dx.doi.org/10.5772/intechopen.91423*

be deeply study the future.

cell proliferation, invasion and migration [48].

and poor prognosis in colon cancer [49].

*2.4.2 In vivo studies of bozepinib*

#### *Bozepinib: A Promising Selective Derivative Targeting Breast Cancer Stem Cells DOI: http://dx.doi.org/10.5772/intechopen.91423*

Mastermind-like MAML-2 protein, which decreases Notch signaling, reducing the primary tumor sphere formation and side population in MCF-7 cell line, contributing to the decrease in the number of CSC subpopulations [42]. We also found an evident downregulation of the *NOTCH 3* gene, which has recently been involved in the proliferation of both HER2 positive and negative breast cancer cells, suggesting that targeted suppression of this signaling pathway may be a promising strategy for the treatment of determined HER2-related breast cancers [43, 44].

The modification of proteins related to the CSC phenotype such as β-catenin, GLI-3, c-MYC and SOX-2 was additionally analysed. This study was carried out in both ALDH+ and ALDH− SKBR-3 breast and HCT-116 colon cancer subpopulations.

β-Catenin is a key mediator of the Wnt signaling which has a crucial role in CSCs [45]. β-Catenin expression was detected in ALDH+ SKBR-3 but not in ALDH− SKBR-3 cells. A high level of expression in HCT-116 cell lines with an ALDH+ subpopulations in comparison with the ALDH− in HCT-116 subpopulations was also detected. After bozepinib treatment, β-catenin expression was reduced in ALDH+ HCT-116 subpopulation. Its inhibition in ALDH− HCT-116 cells was also observed.

GLI-3, a described target gene transcription repressor of Hedgehog signaling pathway [16], was detected in ALDH− HCT-116 isolated cells and was not present in the ALDH+ cells, denoting that the Hedgehog signaling pathway is involved in the CSC phenotype as previously reported [16]. Bozepinib greatly induced the expression of GLI-3 in both ALDH+ and ALDH− HCT-116 cells. However, no changes at protein level were observed in ALDH+/− subpopulations isolated from the SKBR-3 cell line. Furthermore, the activation of GLI-3 protein corresponded with an inactivation of β-catenin expression in CSC subpopulations after bozepinib treatment in accordance with the previous published studies [46]. Considering that GLI-3 overexpression decreased tumor cell proliferation and induced apoptosis in colon CSCs [47], the GLI-3 induction by bozepinib could be one of the mechanisms by which this drug exerts its antitumor activity in colon CSCs. This hypothesis will be deeply study the future.

The c-MYC oncoprotein was observed in SKBR-3 and HCT-116 cell lines with a high level of expression in ALDH+ subpopulations in comparison with the ALDH− subpopulations. Bozepinib induced a significant decrease of c-MYC level in ALDH+ HCT-116 cells and inhibited its expression in ALDH+ SKBR-3-treated cells. It also inhibited ALDH− subpopulations of both HCT-116 and SKBR-3 cells [30]. In accordance with the high level of stem signaling proteins described for CSCs, higher levels of these proteins in the ALDH+ subpopulations in comparison with ALDHcells were detected. The downregulation of c-MYC activates the inhibition of cancer cell proliferation, invasion and migration [48].

The transcription factor SOX2 was detected in HCT-116 ALDH+ subpopulation. However SOX2 was not observed in breast cancer cells, and the expression of SOX2 was very weak in HCT-116 ALDH− subpopulation [30]. Its expression practically disappeared after treatment with bozepinib. SOX2 is involved in the induction and maintenance of pluripotent stem cells and has also been associated with metastases and poor prognosis in colon cancer [49].

#### *2.4.2 In vivo studies of bozepinib*

The acute toxicity profile of bozepinib was determined in BALB/c mice. After 2 weeks, bozepinib was nontoxic to BALB/c mice even at the highest intraperitoneal bolus dose of 200 mg/kg and orally bolus dose of 50 mg/kg. Control mice were treated with vehicle alone. All 50 bozepinib-treated mice remained healthy and gained weight throughout the 15-day observation period, with no evidence of morbidity [28].

*Translational Research in Cancer*

*Therapeutic indexes for 1 and bozepinib.*

*a*

**Table 3.**

**Table 2.**

*determinations.*

*MDA-MB-231.*

enantiomers of some chiral drugs may differ both quantitatively and qualitatively in their biological activity [32, 33]. Moreover, enantiomers can show minimal in vitro but a dramatic in vivo chiral dependency in their antitumor activities [34, 35].

*Anti-proliferative activities of bozepinib and its enantiomers against the cancerous cell lines MCF-7 and* 

*All experiments were conducted in duplicate and gave similar results. The data are means ± SEM of three independent* 

**MCF-7 MDA-MB-231**

**Compound MCF-7 (μM)a MDA-MB-231 (μM)a** Bozepinib 0.355 ± 0.011 0.166 ± 0.063 (*R*)-**2** 0.19 ± 0.001 0.11 ± 0.001 (*S*)-**2** 0.10 ± 0.001 0.11 ± 0.001

**Compound Therapeutic index (TI)**

**1** 4.00 5.50 Bozepinib 5.14 11.0

The cytotoxic effect of bozepinib was determined on SKBR-3 and MDA-MB 468 breast and HCT-116 colon CSC-enriched subpopulations by growing them into low attachment plates with sphere-forming medium for 72 h. Salinomycin is a selective and potent drug used in the treatment of CSCs, but its use is limited in humans due to considerable toxicity [36]. Thus, we decided to include salinomycin in our studies as reference drug. Bozepinib displayed IC50 values in a similar range to salinomycin

CSCs were separated using ALDH activity by FACS. IC50 values were determined in both ALDH positive cells (called ALDH+), ALDH negative cells (called ALDH−) and cells growing in sphere-forming medium without sorter enrichment process [30]. Since there is controversy about the efficacy of sphere-forming cultures to select CSCs [37, 38], we used ALDH activity to enrich cell cultures with clonal ability and stem-like properties. Bozepinib substantially diminished the number and size of spheres at 5 μM and abolished the formation of spheres at 20 μM [30] in subpopulations isolated by ALDH activity. High ALDH activity is associated with metastasis, resistance to chemotherapies and poor prognosis in human cancers, and it has been identified as one of the most specific markers of human CSCs [39]. Apoptosis is also an important factor in the onset of a tumor. It has been reported than CSCs are resistant to apoptosis to ensure succeeding generation [40] and may be on the causes to chemo- or radiotherapy survival. Reactivating death programmes in CSCs may increase efficacy of the treatment. Bozepinib induced a significant level of cell death by apoptosis in the resistant ALDH+ subpopulations from both SKBR-3 and HCT-116 cell lines. Higher levels of apoptosis were detected

Bozepinib induced the downregulation of important genes involved in Notch and Wnt signaling. These genes contribute to the CSC phenotype when they are deregulated [41]. A downregulation was detected of the co-activator

*2.4.1 Effect of bozepinib against CSC subpopulations*

in SKBR-3 and MDA-MB 468 cells [30].

in the ALDH− subpopulations.

**134**

#### *Translational Research in Cancer*

We then evaluated the subacute toxicity after 29 days of intraperitoneal treatment with 100 mg/kg twice a week. Bozepinib-treated mice presented no weight loss or unusual behaviour, and the histopathologic examination did not find any detectable toxicity in the liver or kidneys. These data indicate that, at the concentration used, bozepinib did not cause any systemic damage [30].
