Investigating the effects of two novel 4-MMPB analogs as potent lipoxygenase inhibitors for prostate cancer treatment

Background Lipoxygenases are one of the critical signaling mediators which can be targeted for human prostate cancer (PC) therapy. In this study, 4-methyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine (4-MMPB) and its two analogs, 4-propyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine (4-PMPB) and 4-ethyl-2-(4-methylpiperazinyl)pyrimido[4,5-b]benzothiazine (4-EMPB), were proposed to have anti-tumor properties in prostate cancer. Methods After synthesizing the compounds, cytotoxic effects of 4-MMPB and its two analogs against PC-3 cancerous and HDF normal cells were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and then mechanism of cell death was assessed by flow cytometry. Finally, the anti-tumor effects of the mentioned compounds were investigated in an immunocompromised C57BL/6 mouse model. Results 4-PMPB and 4-EMPB had similar anti-cancer effects on PC-3 cells as compared with 4-MMPB, while they were not effective on normal cells. Moreover, apoptosis and ferroptosis were the main mechanisms of induced cell death in these cancerous cells. Furthermore, in vivo results indicated that both analogs had similar anti-cancer effects as 4-MMPB, leading to delayed tumor growth without any noticeable side effects in weight loss and histological investigations. Conclusion Thus, our results suggest that specific targeting of lipoxygenases via 4-MMPB analogs can be considered as a treatment of choice for PC therapy, although it requires further investigations.

Moreover, it has been demonstrated that HFD results in increased tumor weight and number of intestinal polyps in mice [11,12]. Similar effects have been reported in mouse models with breast cancer [13,14].
Human lipoxygenases (LOXs) are a family of non-heme iron based enzymes that are involved in the metabolism of AA and other PUFAs into biologically active metabolites [15]. So far, several human LOX isoforms including 15-LOX-1, 15-LOX-2, 5-LOX and 12-LOX have been identified and categorized based on enzymatic characterization [16]. For example, 15-LOXs introduce the oxygen at carbon 15 of AA backbone. Two distinct human 15-LOXs are widely distributed in various tissues, exhibiting different substrate preferences. 15-LOX-1, one of the critical enzymes of mammalian LOX family, is physiologically overexpressed in specific cancerous tissues such as prostate [17] and renal cancer [18], while it is poorly expressed in colorectal [19,20], lung [21], esophageal [22] and bladder cancers [23] as well as chronic myeloid leukemia [24]. Thus, 15-LOX-1 is involved in both tumor promotion and suppression depending on the type of cancer. This enzyme predominantly metabolizes linoleic acid (LA) to 13S-hydroperoxyoctadecadienoic acid (13S-HpODE), but also metabolizes AA to 12S-and 15S-hydroperoxyeicosatetraenoic acid (15S-HpETE) [25]. 15-LOX-2 is constitutively expressed in healthy normal prostate tissue [17] while it is down regulated in prostate [26], lung [27] and esophageal cancers [28]. In contrast to 15-LOX-1, 15-LOX-2 preferentially catalyzes AA to 15S-HpETEs and poorly metabolizes LA [29]. Similarly, 5-LOX and 12-LOX utilize AA to synthesize 5S-HpETE and 12S-HpETE, respectively [30,31] and their expression is linked to PC spreading and metastasis [32]. In this context, blocking overexpressed LOX enzymes in PC could be one of the promising targets facilitating drug discovery and treatment. There are several studies reporting different compounds with significant inhibitory effects on 15-LOX-1. Coumarin-based inhibitors, such as 8-farnesyloxycoumarin [33], 5-farnesyloxycoumarin [34] and 7-farnesyloxycoumarin [35] exhibited inhibitory potency against 15-LOX-1 with half maximal inhibitory concentration (IC 50 ) values of 2.64, 8.32 and 11.8 µM, respectively. Furthermore, the inhibitory activities of stylosin and its derivatives have also been shown on soybean LOX-1 [36]. These compounds exerted their anti-cancer effects in PC cells via induction of apoptosis. In addition, indole-based inhibitors, such as 14d [37], indolylpyrazoline (4b) [38] and 9c (i472) [39] are members of another group of potent 15-LOX-1 inhibitors. Sarveswaran et al. [40], treated PC cells with MK591 as a specific chemical inhibitor of 5-LOX activity in order to induce apoptosis via down-regulation of protein kinase C-epsilon (PKCε) and disruption of c-Myc signaling [41]. Moreover, it has been shown that inhibition of 12-LOX activity markedly sensitizes PC cells to radiation both in vivo and in vitro [42,43]. Recent studies have demonstrated that ferroptotic pathway as another type of programmed cell death can be mediated via LOXs in some contexts to eradicate cancers [44,45].

Enzyme inhibitory assessment
Linoleic acid and two assay solutions (A and B) were prepared in advance. Solution A was 50 mM 3-(dimethylamino)benzoic acid (DMAB) in l00 mM phosphate buffer (pH 7.0). Solution B was a mixture of l0 mM 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH; 3 ml), and hemoglobin (5 mg ml −1 , 3 ml) in 50 mM phosphate buffer at pH 5.0 (25 ml). A linoleic acid solution was prepared by mixing 5.6 mg linoleic acid with 0.5 ml methanol and then diluted with 100 mM KOH to a final volume of 5 ml (4 mM). In the assay, a solution of inhibitors in dimethyl sulfoxide (DMSO) (12.5 μl), soybean LOX-1 (4000 units ml −1 in 50 mM phosphate buffer pH 7.0; 25 μl), and phosphate buffer, pH 7.0 (50 mM; 435 μl) were mixed in 48-well plates, and pre incubation was carried out for 10 min at 30 °C. A control test was performed  with the same volume of DMSO. After the pre incubation, linoleic acid solution (25 μl) was added to start the peroxidation reaction at 30 °C, and 10 min later, solution A (135 μl) and then solution B (65 μl) were added to start the color formation. After 3 min, 100 μl of a 2% sodium dodecyl sulphate (SDS) solution was added to terminate the reaction. The absorbance at 598 nm was compared with the control. These experiments were performed in triplicate and the data were analyzed using GraphPad Prism 6.0 (San Diego, USA).

Preparing different solutions of compounds for cell culture
Two mg of each compound was dissolved in 100 μl hydrochloric acid (HCl 0.3 N) to make a primary stock solution. Different concentrations (1.56-50 μg ml −1 ) were then prepared by serial dilution from the primary stock solution and further diluted in the culture medium. Moreover, culture medium containing 0.25% HCl 0.3 N was used as a negative control in all experiments for a better evaluation.

Cell culture
PC-3 cells were obtained from Pasteur Institute (Tehran, Iran) and routinely cultured in RPMI 1640 supplemented with 10% FBS. In order to investigate the toxicity of the compounds on normal cells, in vitro experiments were also performed on HDF cells. These cells were kindly provided by Academic Center for Education, Culture and Research (ACECR, Mashhad, Iran) and grown in DMEM supplemented with 10% FBS. Both cell lines were kept at 37 °C containing 5% CO 2 in a humidified incubator.

Cytotoxicity assay
The MTT reduction assay is a standard quantitative method to evaluate cytotoxicity. This assay measures cellular dehydrogenases, and is considered as an easy, safe and highly reproducible method. Viable cells reduce yellow tetrazolium salt to purple formazan crystals and thus the color change serves as a marker reflecting cell viability [48]. PC-3 and HDF cells were seeded in 96-well plates (NEST, China) at a density of 6000 and 8000 cells per well, respectively and incubated at 37 °C with 5% CO 2 . Next day, the media were changed and both cell lines were treated with various concentrations (1.56-50 μg ml −1 ) of the compounds and cisplatin. Experiments were carried out in triplicate for 4-PMPB, 4-EMPB and 4-MMPB as well as cisplatin as a positive control. After 24, 48 and 72 h of treatments, 20 μl MTT solution (5 mg ml −1 in PBS; phosphate buffered saline) was added to each well, and cells were incubated in the dark for 3-4 h. The media were then removed and purple crystals were dissolved in 160 μl DMSO. The absorbance in each well was measured at 540 nm as reference wavelength in an enzymelinked immunosorbent assay (ELISA) reader (Awareness Technology, USA) and relative optical density (OD) values were recorded. The percentage of viable cells was calculated using the following equation [49]:

Flow cytometric analysis
To determine the mechanism of cell death induced by 4-MMPB analogs, flow cytometry was employed using the Annexin V-FITC kit with PI (BioLegend, San Diego, USA) according to the manufacturer's instructions. We performed co-treatment experiments with the compounds and a ferroptosis inhibitor (IC 50 values of 4-PMPB + , 4-EMPB + , and 4-MMPB + deferoxamine (80 µM)) on PC-3 cells, to distinguish between ferroptosis and apoptosis mechanisms [50]. In order to evaluate the apoptosis mechanism, cells were treated with the ½ × IC 50 and IC 50 values of 4-PMPB, 4-EMPB, 4-MMPB, and cisplatin for 24 h. Then the cells were collected by trypsinization (0.25% trypsin-1 mM EDTA), washed twice with PBS, followed by centrifugation, and resuspended in a binding buffer. Samples were stained with Annexin V-FITC/ PI followed by 15 min incubation at room temperature. Finally, cells were subjected to flow cytometry (BD Accuri C6, San Jose, USA) and the data were analyzed by FlowJo 7.6.1 software (Ashland, USA). Moreover, PC-3 cells treated with 0.3 N HCl were used as a control to eliminate the effects of solvent.

Anti-cancer evaluation of compounds on a PC-3 xenograft tumor model
The and maintained under pathogen-free conditions. Immunosuppression of male C57BL/6 mice (4-6 weeks old) was carried out according to the protocol described by Jivrajani et al. [51]. However, in this study, for immunosuppression ketoconazole and ampoxin drugs were replaced with itraconazole and co-amoxiclav. After completion of this procedure, the total white blood cell (WBC) and lymphocyte counts were evaluated to confirm the immunosuppression protocol.

Statistical analysis
All statistical analyses were carried out using GraphPad Prism software 6.0 (San Diego, USA). First the Kolmogorov-Smirnov test was performed to evaluate the normal distribution of the data. Then, differences between groups were assessed with Student's t-test (group pairs) or one-way ANOVA (multiple groups) followed by Tukey multiple comparison tests. Experiments were performed in triplicate and data are presented as mean ± standard error or standard deviation as required. p-values < 0.05 were considered as significant.

In vitro cell viability assay
In present study, we first evaluated in vitro cytotoxicity and anti-cancer properties of 4-PMPB and 4-EMPB on PC-3 and HDF cells, and the results were compared with 4-MMPB as a potent 15-lipoxygenase inhibitor and cisplatin as a well-known chemotherapeutic drug. After exposing both cell lines to different concentrations of compounds, cell viability was determined by MTT assay and IC 50 values were calculated. The IC 50 values and dose-response curves are presented in Table 2 and Fig. 3, respectively. Dose-response curves indicated that 4-PMPB and 4-EMPB have similar anti-cancer effects on PC-3 cells as compared with 4-MMPB. As shown in Table 2, 4-PMPB, showed no obvious cytotoxicity on normal cells, while, 4-EMPB and 4-MMPB exhibited a weak cytotoxicity on HDF cells. Furthermore, cisplatin showed prominent cytotoxic effects on both prostate cancer and normal cells.

Evaluating the mechanism of cell death
To evaluate the mechanism of cell death, PC-3 cells cotreated with 4-PMPB, 4-EMPB, or 4-MMPB in combination with deferoxamine, a ferroptosis inhibitor, were assessed using Annexin V-FITC/ PI staining and compared with those treated with the compounds only. The results revealed that the population in Q2 (Annexin V positive/PI positive) was markedly decreased after cotreatment with deferoxamine as compared to those evaluated in the absence of this ferroptosis inhibitor. These observations suggest that 4-MMPB and its two analogs have probably induced both apoptosis and ferroptosis in PC-3 cells (Fig. 4). Flow cytometry-based quantification showed the percentage of early and late apoptotic/ ferroptotic cells (Q3 + Q2) increased from 0.133% in control to 52.38%, 60.9%, 41.5% and 67.8% in 4-PMPB-, 4-EMPB-, 4-MMPB-, and cisplatin-treated PC-3 cells, respectively, when treated with IC 50 values of the compounds.

In vivo anti-tumor effects of the compounds
After administration of immunosuppression protocol, blood samples were collected and their parameters were compared with normal C57BL/6 mice. Significant reduction in WBC, lymphocyte, and neutrophil counts (p < 0.0001) was confirmed in immunocompromised mice (Fig. 5). On day 11 after immunosuppression, PC-3 cells were injected subcutaneously. Immunocompromised mice bearing PC-3 tumors were then treated with PBS, 0.3 N HCl, 4-PMPB (10 and 50 mg kg −1 ), 4-EMPB (10 and 50 mg kg −1 ), 4-MMPB (10 and 50 mg kg −1 ), and cisplatin, and then their body weights and tumor volumes were measured every other day. As shown in Fig. 6a-c, tumor growth was effectively inhibited after treatment with high doses of 4-PMPB, 4-EMPB, and 4-MMPB. In contrast, tumors in control groups receiving PBS and 0.3 N HCl grew markedly during the 15 days follow up period. Furthermore, the positive control group, receiving cisplatin, exhibited a significant decrease in tumor growth as compared with the controls and other experimental groups with lower doses. The results revealed that, there was no significant difference between tested compounds (4-PMPB, 4-EMPB) and 4-MMPB in tumor inhibition.

In vivo side effects evaluation
The side effects of the compounds were evaluated by measuring the body weights, hematological analyses, and H & E staining of different organs. Body weights of various groups were compared and as shown in Fig. 6d and e, the cisplatin treated group demonstrated the lowest body weights, which were significantly lower than other experimental groups (p < 0.01). However, no significant changes in body weights were observed between 4-PMPB-(10 and 50 mg kg −1 ), 4-EMPB-(10 and 50 mg kg −1 ) and 4-MMPB-(10 and 50 mg kg −1 ) treated groups.
To determine possible side effects of various treatments in vivo, biochemical parameters such as urea, aspartate transaminase (AST) and alanine aminotransferase (ALT) were evaluated in different experimental groups (Fig. 7). Urea levels did not change in all treatment groups as compared with the immunosuppressed group. Mice treated with 4-PMPB, 4-EMPB and 4-MMPB at concentrations of 50 mg kg −1 had significantly elevated AST and ALT levels compared with immunosuppressed control group. Similarly, the group treated with cisplatin had significantly increased serum liver enzymes compared to control group (p < 0.0001). Histological analysis showed no obvious tissue damages in major organs including liver, kidney, and spleen, following 4-PMPB, 4-EMPB, and 4-MMPB treatments. However, mild and local accumulation of inflammatory cells was noticed in cisplatin treated group (Fig. 8). Meanwhile, the tumor tissue treated with cisplatin and high doses of compounds exhibited a higher degree of cell death than the other experimental groups (Fig. 9). Apoptosis of tumor cells with nuclear and cytoplasmic condensation and also tumor necrosis were visualized in treated groups. However, further immunohistochemical analyses are required to confirm the exact type of cell death in tumor tissues.

Discussion
In recent years, cancer incidence rate has increased significantly especially in developing countries and discovering more effective treatments has emerged as a major concern. Chemotherapy is one of the conventional treatment modalities, which can increase patient's survival. However, therapeutic index can be decreased due to serious limitations of chemotherapy including drug resistance, poor specificity, and undesirable, long term side effects [53,54]. Discovering cell signaling molecules which can trigger an apoptotic pathway in cancerous cells is crucial to improve therapeutic outcomes. Lipoxygenases are considered as a family of important signaling mediators that their overexpression is associated with tumor initiation and progression of prostate cancer [55]. Development of effective lipoxygenase inhibitors may successfully diminish cell proliferation in the tumor mass. An example of established 15-lipoxygenase inhibitors is 4-MMPB [46], which has been well documented in recent studies on PC-3 cells [33][34][35][36]. In this study, two 4-MMPB analogs, 4-PMPB and 4-EMPB, with inhibitory effects on lipoxygenase activity [46,47] were employed and 4-PMPB indicated the most selective anti-proliferative effects as compared with the other two. The significantly lower cytotoxicity of these compounds on HDF cells can be attributed to the low expression of lipoxygenases in these normal cells [56,57]. Considering the lower levels of cell death population after co-treatment with 4-PMPB, 4-EMPB or 4-MMPB compounds and deferoxamine, compared with those treated with the compounds only, it is proposed that cell death is triggered through both apoptosis and ferroptosis pathways. It has been shown that ALOX15 expression is regulated by a mutant form of P53 and results in promoting PC [58]. Moreover, 13S-HODE, the major metabolite of 15-LOX-1 can interact with anti-apoptotic mediators such as B-cell lymphoma 2 (BCL2) and insulin-like growth factor receptor-1 (IGF-1R) which leads to inhibition of apoptotic signaling pathways in PC [59][60][61]. Additionally, recent studies demonstrated that 15-LOX-1 is one of the fundamental regulators in ferroptosis pathway  [44,62], and is involved in PUFA oxidation and pro-ferroptotic lipid peroxide generation [63]. In order to better evaluate the efficacy of anti-cancer agents, treatment strategy should also be assessed in animal experimental models [64,65]. Nude and immunocompromised mice models are commonly used as hosts for human cancer cell lines. However, nude mice bear several drawbacks due to high cost, low tumorigenesis rate, high sensitivity, difficult maintenance and high mortality rate [66]; therefore we used immunocompromised mice bearing human PC as a preferred model. There are a number of immunosuppressive medications such as glucocorticoids, azathioprine, cyclophosphamide, methotrexate, cyclosporine, ketoconazole and tacrolimus which are used alone or in combination to prevent human tumor xenografts rejection in murine models [64]. Cunha et al. [67] reported xenotransplantation of human glioblastoma cells in rats by cyclosporine treatment (5 mg per kg per day) until the end of experiments. Similarly, Du et al. [68] reported use of cyclosporine treatment (10 mg per kg per day) for development of human hepatocarcinoma cells xenograft in a C57BL/6 mouse model. Moreover, Rose et al. [69] reported that xenologous skin transplantation in an immunosuppressed sheep model was made possible by using cyclosporine (2-3 mg kg −1 ) in combination with ketoconazole (10 mg kg −1 ) after 18 days. In present study, human PC xenograft was developed according to the Jivrajani's protocol in C57BL/6 mice [51]. Blood count analysis after administration of immunosuppression protocol, and tumor growth volume in control groups confirmed successful immunosuppression and xenotransplantation in mouse model. Moreover, in vivo results demonstrated that while cisplatin treatment led to a significant decrease in tumor size, it suffered from side effects including a significant decrease in body weight. Furthermore, the highest dose of 4-PMPB and 4-EMPB (50 mgkg −1 ) possessed similar anti-cancer effects as compared with 4-MMPB, leading to delayed tumor growth without noticeable side effects in weight loss and histological assessments.

Conclusion
Targeted therapeutic strategies such as lipoxygenase inhibition in PC can reduce tumor growth and help in improving clinical outcomes. Data generated from our study showed that, use of 4-PMPB and 4-EMPB can be considered as a treatment of choice to trigger both apoptosis and ferroptosis pathways and inhibit PC cell survival in vitro and in vivo probably via specific targeting of lipoxygenases. For this purpose, different approaches can be recommended, and use of nanotechnology-based drug delivery systems (DDSs) is one of the most attractive strategies. In this regard, mentioned compound inhibitors of lipoxygenases can be encapsulated into specific