The effect of “NutramilTM Complex,” food for special medical purpose, on breast and prostate carcinoma cells

NutramilTM Complex is a multicomponent food product that meets the requirements of a food for special medical purpose. As a complete, high-energy diet it consists of properly balanced nutrients, vitamins and minerals. The aim of this study was to assess the effect of NutramilTM Complex on breast and prostate carcinoma cells. Our results showed that NutramilTM Complex reduced the viability and proliferation of breast and prostate cancer cells and that this process was associated with the induction of apoptosis via activation of caspase signalling. Data showed elevated levels of p53 tumour suppressor, up-regulation of p38 MAPK and SAPK / JNK proteins and downregulation of anti-apoptotic ERK1/2, AKT1 and HSP27. Treatment with NutramilTM Complex also affected the expression of the BCL2 family genes. Results also showed down-regulation of anti-apoptotic BCL-2 and up-regulation of pro-apoptotic members such as BAX, BAD, BID. In addition, we also observed regulation of many other genes, including Iκβα, Chk1 and Chk2, associated with apoptotic events. Taken together, our results suggest activation of the mitochondrial apoptotic pathway as most likely mechanism of anti-carcinogenic activity of NutramilTM Complex.


Introduction
Breast cancer is one of the most common type of cancer affecting women around the world [1], and is also the leading cause of cancer death in the female part of the population [2]. Prostate cancer, on the other hand, occupies the third place of type of cancer for males, and fourth place as the cause of cancer deaths [3]. Although improving diagnostic tools helps with the patients' outcome, research in cancer prevention remains insufficient.
Numerous studies showed that selected nutrients and non-nutrients can act preventively in cancer. They included, among others, diets rich in vegetables (i.e. cruciferous vegetables, tomatoes), fruits, oily sea fish and soybeans; thus, rich in vitamin E, C, carotenoids (beta-carotene, lycopene) selenium, omega-3 fatty acids, glucosinolates, polyphenols and etc. [4][5][6]. PLOS  Detection was executed by chemiluminescence, using Clarity™ Western ECL Substrate (Bio-Rad, CA, USA). Western blot stripping buffer (Thermo Scientific, MA, USA) was used to remove the antibodies from the membrane.

Statistical analysis
All experiments were performed in at least three independent experiments and measured in triplicates. Shapiro-Wilk's test was applied to assess normality of distribution. An independent samples t-test was applied to compare unpaired means between two groups and P 0.05 was considered statistically significant. All analyses were performed using Statistica ver.12 (Stat-Soft, Tulsa, OK, USA).

Cytotoxicity
Nutramil TM Complex showed a cytotoxic effect on all examined cells, both cancer and nonmalignant in a dose-and time-dependent manner ( Table 1). Cytotoxicity levels for NC-CC were lower than those observed for NC (Table 1). All cell lines showed mostly necrotic changes at 10% concentration of NC ( Table 1). The cytotoxicity results for treatment with 5% NC was

Cell viability
Treatment with 4% Nutramil TM Complex decreased the viability of MCF-7 breast cancer cell line by 20% after 24-48 h and by 35% after 72 h (P 0,001; Fig 1A). Results for NC-CC showed a similar trend, while CC showed quite the opposite-significant increase in the MCF-7 cells viability (P 0.001; Fig 1A). Treatment with 4% Nutramil TM Complex also decreased the viability of prostate cancer cells by 30% after 48 h and by 70% after 72 h (P<0,001, Fig 1B and 1C). The results did not differ significantly between androgen-independent DU145 cell line and androgen-dependent LNCaP. On the other hand, NC treatment increased significantly the viability of the normal prostate cells PNT-2 after 24-48 h to 150% of the UC. Interestingly, after 72 h viability sharply decreased to 60% of UC (P 0.001; Fig 1D).
DU145 and LNCaP cells treated with 4% NC-CC showed a significant, 30-40% reduction in viability (P 0.001; Fig 1B and 1C). Viability of PNT-2 cells was not affected by NC-CC after   Fig 1D). Treatment with calcium caseinate cells showed a significant increase in cell viability across all tested cell lines (Fig 1A-1D).

Cell proliferation
BrDU labeling results showed that Nutramil TM Complex reduced the proliferation of studied cell lines in a dose-and time-dependent manner. For MCF-7 breast cancer cells, proliferation was reduced by approximately 30% at 48-72 h (P 0.001, Fig 2A). Similar trend was observed for the hormone-independent DU145 prostate cancer cell line, where proliferation decreased by 40% at 72 h post-treatment ( Fig 2B). Most prominent were results for LNCaP (androgendependent) prostate cancer cells that showed the highest level of susceptibility to NC in our study. Reduction in proliferation by 30% was observed already at 24-48 h and reached 40-45% at 72 h post-treatment ( Fig 2C). Interestingly, PNT-2 normal prostate cells reacted similarly to cancer cells and showed approximately 30-40% reduction in proliferation after treatment with NC ( Fig 2D).
Results for NC-CC showed a similar trend to the NC treatment (Fig 2A-2C). Almost 30-35% reduction in proliferation for MCF-7 and DU145 cell line (P 0.001; Fig 2A and 2B) and about 40% reduction after 72 h post-treatment for LNCaP cell line (P 0.001; Fig 2C). On the  other hand, proliferation of PNT-2 normal cell line significantly increased with time of treatment (P 0.001; Fig 2D).
Treatment with CC did not show statistically different results for proliferation of MCF-7 and PNT-2 cells vs UC (Fig 2A and 2D); however, it did not promote proliferation of prostate cancer cells (Fig 2B and 2C).

mRNA expression of genes associated with cell cycle and apoptosis
To further evaluate the effect of Nutramil TM Complex on studied cancer cell lines, we investigated its effect on the expression of genes associated with cell cycle and apoptosis. Our analysis showed that treatment with NC has an effect on mRNA levels of multiple genes. All results are presented in Table 2. For MCF-7 breast cancer cells, data showed a significant decrease in the mRNA levels of BCL-2, KRAS and NRAS oncogenes and some of the YWHA family. Pro-apoptotic genes, including APAF1, CASP8, BID, BAD, BAX, and TP53 were up-regulated.
Similar trends were observed for treatment with NC-CC for all examined cell lines.

Expression of proteins involved in cellular stress and apoptosis signaling
PathScan1 Stress and Apoptosis Signaling Antibody Array analysis was performed for MCF-7 ( Fig 3A and 3B) and DU145 (Fig 4A and 4B) to further investigate the effect of Nutramil TM Complex on proteins involved in cellular stress, cell cycle and apoptosis signaling.
In addition, levels of Cytochrome c, Smac/Diablo and HtrA2/Omi mitochondrial proteins were measured (Fig 5A and 5B).
For MCF-7 breast cancer cells, Nutramil TM Complex reduced significantly the expression of pro-survival AKT1 down to 82% of UC (P 0.05; Fig 3B). The same trend was found for the NC-CC treated cells (68% of UC, P 0.05; Fig 3B). For pro-survival HSP27, results showed a significant decrease in protein expression only for NC treated cells (60% of UC; P 0.05; Fig  3A). No changes were measured for Survivin ( Fig 3B). Results for TAK1 showed a significant increase in the level of active protein form for both NC and NC-CC treated cells (209% and 194% of UC, respectively; P 0.01; Fig 3A). Nutramil TM Complex had a significant effect on the p38 MAPK level (183% of UC; P 0.05; Fig 3A) as well as on SAPK/JNK MAP (127% of UC; P 0.01; Fig 3A). Similar effects for both p38 MAPK and SAPK/JNK MAP were observed for NC-CC (152% of UC and 161% of UC respectively; P 0.01; Fig 3A). Significant increase of total Iκβα was measured for both NC and NC-CC treated MCF-7 cells (309% of UC and 288% of UC; P 0.01; Fig 3A). However, results for NC showed a significant reduction in levels of the phosphorylated form of Iκβα to 72% of NC (P 0.05; Fig 3A). EIF-2α is the protein required to initiate translation processes. Our results showed a slight increase in phosphorylated protein form in cells treated with both NC and NC-CC (both 121% of UC; P 0.05; Fig  3A).
Our results showed strong activation of pro-apoptotic proteins in MCF-7 cells treated by Nutramil TM Complex, including p53 (128% of UC; P 0.05; Fig 3B). Caspase-3 and Caspase-7 cleavage was significantly increased after NC treatment (177% and 150% of UC, respectively P 0.05; Fig 3B). These results were also statistically significant for NC-CC, for which Caspase -3 protein expression levels were 200% of UC (P 0.01; Fig 3B). In addition, results showed an increase in cleaved form of PARP after both NC and NC-C treatment (146% of UC and 124% of UC, P 0.01, respectively; Fig 3B). Our results also showed an increase in protein levels of Cytochrome c, Smac/Diablo and HtrA2/Omi (Fig 5A), which promote caspases activity. The effect of NC-CC on the expression of apoptosis markers was similar to that of NC ( Fig 5A), with the exception of HtrA2/Omi (Fig 5A). Chk1 and Chk2 kinases play an important role in DNA damage checkpoint control. Results for both showed a significant increase in protein expression levels after NC treatment (150% of UC and 237% of UC; P 0.01; Fig 3B). Similar trend was observed for NC-CC treated cells (113% of UC and 230% of UC, respectively Fig  3B). For DU145 cells, treatment with Nutramil TM Complex reduced the level of pro-survival proteins such as ERK1/2 to 63% of UC (P 0.05; Fig 4A), AKT1 to 70% of UC (P 0.05; Fig  4B), HSP27 to 83% of UC (NS; Fig 4A), Survivin to 78% of UC (P 0.01; Fig 4B), phospho-BAD to 90% of UC (NS; Fig 4B) and phospho-Iκβα to 81% of UC (P 0.05; Fig 4A). Expression of TAK1 was increased to 204% of UC (P 0.05; Fig 4A). On the other hand, the expression of pro-apoptotic proteins, p53 and SMAD-2, was increased (124% and 134% of UC, respectively; P 0.05; Fig 4B). Caspase-3 and Caspase-7 cleavage was found increased to 167% and 150% of UC respectively (P 0.01; Fig 4B). In addition, results also showed increased levels of cleaved PARP cleavage (250% of UC; P 0.01; Fig 4B) as well as mitochondrial Cytochrome Effect of "Nutramil TM Complex" on breast and prostate carcinoma cells Effect of "Nutramil TM Complex" on breast and prostate carcinoma cells c and HtrA2/Omi (Fig 5B). Total Iκβα increased to 158% of UC (P 0.05; Fig 4A), p38 MAPK to 146% of UC (P 0.05; Fig 4A) and SAPK/JNK to 130% of UC (P 0.05; Fig 3B). Significant increase in protein expression was found for Chk2 (272% of UC; P 0.01; Fig 4A); however, NC had no measurable effect on Chk1 level (99% of UC; Fig 4B). EIF-2α protein expression was 119% of UC after NC treatment (P 0.05; Fig 4A).

Discussion
Nutramil TM Complex is a complete nutritional supplement, providing about 417 kcal/100 g from 60% carbohydrate, 25% fat and 15% protein. It was designed to contain all essential nutrients i.e. basic nutrients, minerals and vitamins in balanced proportions as required by dietary recommendations. The high-quality protein is delivered in the form of calcium caseinate, which is a milk protein. Carbohydrates, including sugars, are supplied in form of maize maltodextrin with varying absorption rates. The product is clinically free of lactose. As sources of fats include: rapeseed oil (80%) and medium-chain triglycerides (MCTs) (20%). It provides very important mono-unsaturated oleic acid, essential fatty acids, as well as long-chain triglycerides (LCTs). MCTs provide fast and lasting energy and are easily absorbed by the body. In addition, they also have a positive effect on calcium absorption.
In our previous pilot studies, we have shown that Nutramil TM Complex decreases the viability of breast and prostate cancer cells [7,8]. In current manuscript, we made an attempt to determine the potential molecular mechanism after treatment with NC (Figs 1A-1C and 2A-2C). To examine whether the apoptosis was the primary cause of cell death, we analysed the levels of genes associated with apoptotic events. Further, we verified the effect of NC on selected proteins involved in cellular stress signalling related to apoptosis induction (Fig 6).
Our results for NC treated cancer cells showed an increased expression of TP53 in all examined cell lines. We also measured promoted expression of CDKN2A in prostate cancer lines ( Table 2). The TP53 gene is a key cancer suppressor gene. The p53 protein controls the transcription of many different genes in response to stress signals, thus regulating processes related to DNA repair, cell-cycle arrest or apoptosis. In turn, the CDKN2A codes for p16 and p14ARF proteins that act via two independent pathways: retinoblastoma RB-and p53-tumour suppressor pathways, respectively [10]. Chk1 and Chk2 play an important role in DNA damage checkpoint control (Fig 6). They also affect the post-translation modifications of p53 protein, leading to its accumulation [11]. Our results confirm over-expression of p53 protein after NC treatment (Figs 3B and 4B) as well as an increase in Chk1 and Chk2 protein expression (Figs 3B and 4B). As a transcription factor, p53 is responsible, among others, for the regulation of mitochondrial-induced pro-apoptotic proteins of BCL-2 family [12,13]. BCL-2 is a family of proteins regulating the apoptosis processes by controlling the mitochondrial permeability [14]. Our results showed that NC increased gene expression of pro-apoptotic BAX, BID, BAD members, and decreased expression of anti-apoptotic BCL-2 ( Table 2). In addition, we determined up-regulation of APAF1 and CYCS genes (Table 2), which encodes a cytoplasmic protein that initiates apoptosis (Fig 5). Apaf-1 protein is a caspase-activating molecule that is released from the mitochondria during apoptosis induction. Activation of p53 results in oligomerization of pro-apoptotic members (BAX, BID, PUMA, NOXA) at the mitochondrial outer membrane and translocation of Cytochrome c and Apaf-1 into cytosol followed by activation of Caspases [15]. The Caspase-3 and Caspase-7 are the downstream effector caspases that cleave specific cellular targets. PARP is one of the earliest nuclear enzymes to be targeted by Caspase-3 during apoptosis [16]. In our study, we confirmed the activation of pro-apoptotic markers (Cytochrom c, Smac/Diablo, HtrA2/Omi) as well as Caspase-3 and 7 under NC (Figs 3B, 4B, 5A and 5B). We also measured an increased level of cleaved form of PARP (Figs 3B and 4B).
In our work, we also verified the impact of NC on selected proteins involved in cellular stress response and their links to apoptosis induction (Fig 6). Our results showed that NC treatment decreased expression of ERK1/2 ( Fig 4A) and AKT1 (Fig 4B) proteins in prostate cancer cells and AKT1 and HSP27 cells in MCF-7 cells (Fig 3A and 3B). AKT kinase is a major signaling node that transmit signals related to many essential pro-survival cellular functions. The main consequence of its activation is increased proliferation and tumor transformation and inhibition of apoptosis [17,18]. In addition, AKT phosphorylated the pro-apoptotic BAD protein at Ser136 and inhibits its ability to induce apoptosis [19,20]. HSP27 is a mediator of Effect of "Nutramil TM Complex" on breast and prostate carcinoma cells cellular stress that confers resistance to adverse environmental conditions. It was associated with the inhibition of Cytochrome c release from mitochondria and with an increase in AKT expression [21].
Our results showed a significantly elevated level of phosphorylated p38 MAPK and SAPK/ JNK MAP kinases in both MCF-7 and DU145 cell lines after the treatment with NC (Figs 3A  and 4A). Activation of p38 MAPK and SAPK/JNK MAP kinases occurs via dual phosphorylation mechanism in response to cellular stressors and leads to regulation of the inflammatory response, cell-cycle arrest and apoptosis induction. It has been suggested that SAPK/JNK can contribute to the both, the c-Jun / AP-1 external apoptosis pathway as well as to the mitochondria-dependent apoptosis pathway via p53 and pro-apoptotic BCL-2 family activation. In addition, the suggestion was made that these mechanisms can act independently or co-operate with one another to induce cell death [22]. Our results give support to this hypothesis as they showed an increased expression of FADD and CASP 8 genes in both breast and prostate cancer cell lines and for the prostate cell lines also of FAS gene, associated with the external apoptosis pathway ( Table 2).
Our results showed also an increase in MYC mRNA expression in both MCF-7 and DU145 lines, while its down-regulation in LNCaP (Table 2). MYC expression is closely associated with the progression of cell-cycle in normal tissues and its overexpression has been found in variety of cancer types. Interestingly, increasing number of research shows that MYC can be also involved in the control of apoptosis. McMahon [15] in his review points out that for normal cells when growth factors are limiting, cells with high MYC levels may activate p53 protein, leading to the induction of apoptotic events. However, in the absence of p53 activation and / or pre-dominance of pro-life factors such as BCL-2, high MYC expression may be insufficient to trigger apoptosis. Our results, demonstrated overexpression of MYC, p53 accumulation as well as lowered levels of BCL-2 expression, supporting this hypothesis.
In our study, we also investigated the effect of NC on the activity of Iκβα. Iκβα protein regulates the activity of NF-κβ transcription factor by forming a protein complex that prevents translocation of NF-κβ to the nucleus. Presence of phosphorylated form of Iκβα indicates the degradation of the NF-κβ / Iκβα complex and translocation of NF-κβ into the nucleus, where as a transcription factor it can activate many pro-or anti-apoptotic genes [23]. Treatment with Nutramil TM Complex significantly increased the level of un-phosphorylated Iκβα, capable of binding to NF-κβ (Figs 3A and 4A). Interestingly, our results showed elevated level of TAK1 protein, which may promote phosphorylation of Iκβα protein [24] while measuring lower levels of phosphorylated Iκβα after NC treatment (Figs 3A and 4A).
Many studies are available on individual components of Nutramil TM Complex that show their potential anti-cancer properties. For MCT fats, they have been shown to delay tumour growth in a mouse xenograft model [25] and to exacerbate the therapeutic effect of studied anti-cancer substances in TCC cells in dogs [26]. Similarly, rape seeds have been shown to exhibit very strong antioxidant activity and have the potential to inhibit the proliferation of tumour cells [27,28]. Studies in MCF-7 and T47D breast lines showed their inhibitory effect on cancer cell growth leading to induction of apoptosis via increased expression of Caspase-3 and p53 [29]. In the in vivo studies, canola oil diet significantly reduced the incidence as well as multiplicity of colorectal tumours in rats [30].
Nutramil TM Complex contains all vitamins and minerals, including macronutrients and micronutrients essential for proper functioning of the human body (S1 Table). Some of them, including vitamin D, C, K, E, B complex vitamins and minerals, i.e. selenium, zinc and iodine, have proven anti-tumour activity [31][32][33]. Deeb et al. [34] have demonstrated that the active form of vitamin D3 (1α, 25 (OH) 2D3) exhibits anti-tumour properties by regulating the expression of Bcl-2 family proteins and by activating the Caspases. Vitamin D3 in Nutramil TM is in the form of cholecalciferol, thus the pro-apoptotic effect of 1α,25 (OH)2D3 may not be directly translated into the observed properties of the formulation; however, some studies suggest that cholecalciferol may be metabolized in some tumour cells, especially in breast cancer cells [35]. Studies in vitro on lymphoma cells using ascorbic acid (form of vitamin C present in Nutramil TM Complex) showed a decrease in the viability of cancer cells without affecting normal cells [36]. In turn, vitamin K affects the levels of tyrosine kinases, phosphatases and activation of transcription factors Myc and Fos, followed by the regulation of expression of genes involved in cell cycle regulation and apoptosis induction [37].
For B complex vitamins, deficiencies in B 6 and B 12 can cause DNA damage and lead to cancer [38,39]. Riboflavin reduces the risk of cancer by acting as a cofactor in folate metabolism [40] and by enhancing the anti-cancer activity of vitamin C [41]. The effect of thiamine supplementation is not yet fully understood. On the one hand, it has been shown that thiamine can enhance cancer cell proliferation and increase therapeutic resistance [42,43]; on the other hand, a high dose of this vitamin has been shown to have an inhibitory effect on tumour cell growth [42,44]. Vitamin E includes a vast group of compounds belonging to the tocopherols and tocotrienols family, which have been shown to have anti-tumour properties [45]. It has been shown that some vitamin E components such as RRR-alpha-tocopheryl succinate and tocopherol ether may have pro-apoptotic effect on cancer cells in a dose that does not affect healthy cells [46]. However, there are no reports linking the DL-α-tocopheryl acetate, which is an active vitamin E form in the Nutramil TM Complex, with reduced survival of cancer cells. Another component of Nutramil TM Complex is the sodium selenite, which anti-tumor properties have been demonstrated in animals [47] and in cell lines including fibrosarcoma, lung carcinoma and acute promyelocytic leukemia [48][49][50][51]. One of in vivo studies involved nude mice bearing human colorectal carcinoma SW480 cell line xenografts, which were injected peritoneally for 21 days with sodium selenite. Results showed significant tumour suppression. In addition, authors showed reduced expression of Bcl-xL proteins and increased expression of proapoptotic proteins, including Bax, Bad and Bim as well as Caspase-9 [47]. In studies using U2OS cells, induction of apoptosis has been shown to be associated with activation of Caspase-3, up-regulation of TP53 and PTEN suppressors and down-regulation of BCl-2 expression. In promyelocytic leukemia NB4 cells, sodium selenite induced reactive oxygen species generation that was associated with early events that triggers endoplasmic reticulum stress mitochondrial apoptotic pathways [51]. Finally, for A549 human lung carcinoma cells, sodium selenite has been shown to modulate both extrinsic and intrinsic apoptotic pathways. Apoptosis induction was mediated by reactive oxygen species (ROS) and was dependent on the activation of Caspases [49].
Zinc whose anti-neoplastic properties have been described for breast cancer cells and prostate cancer [52] is also present in the formulation. In both cell lines, apoptosis was associated with the mitochondrial pathway, including events such as activation of pro-apoptotic BAX, Cytochrome c translocation and Caspase activation. In breast cancer cells (MCF-7) induction of apoptosis was accompanied by accumulation of p53 protein [53], whereas in PC-3 prostate cancer cells, apoptosis was independent of p53, but also via the mitochondrial pathway [52,54]. Another mineral ingredient of the Nutramil TM formulation with potential anti-neoplastic significance is magnesium. This element participates in many metabolic processes and redox reactions [55]. Magnesium present in drinking water was associated with reduction of the risk of liver cancer [56], while high-dose magnesium supplementation was shown to reduce the risk of colorectal cancer [57]. Epidemiological studies confirm the relationship between magnesium deficiency and colorectal cancer in overweight people [58]. It is also worth mentioning the role of calcium ions in regulation of proliferation and induction of apoptosis. Regulation of calcium ion concentration is dependent on activation of ion channels, including K + and Clchannels, that regulate the membrane potential and increase the probability of Ca 2+ channel opening. Also, the activity of individual ions affects their concentration in the cytoplasm [59]. The correlation has been shown between deregulation of these ions and apoptosis. Increasing intracellular chloride and sodium ion concentrations results in increased ROS, release of Cytochrome c into the cytoplasm and cell apoptosis [60]. Finally, among the listed mineral components of the preparation, iodine also has been shown to have a pro-apoptotic activity on breast cancer (MCF-7) [61].
In conclusion, our study, as one of very few, show the effect of complete food medical supplement on cancer cells. It is well known that cells integrate multiple signals from a variety of sources before following either pro-or anti-apoptotic pathway. Taking into account the significant reduction if cancer cell growth (Figs 1A-1C and 2A-2C) with simultaneous activation of many pro-apoptotic proteins, tumour suppressors and regulators of cell-cycle (Figs 3A, 3B, 4A. 4B, 5A and 4B and Table 2), it can be concluded that Nutramil TM Complex exhibits anti-tumor properties. Moreover, our data suggest that it activates apoptotic events via mitochondrial-induced pathway (Fig 6). This is most likely related to the composition of the preparation: the optimum proportion, bioavailability and cellular metabolism of the individual components. It appears that Nutramil TM Complex as a Food for Special Medical Purpose can support the treatment of oncological patients, not only due to their complete nutritional value, but also for the cytotoxic effects on tumour cells. We admit that our results are based on the in vitro model which requires further verification in vivo; However, according to the 3R principles (Replacement, Reduction and Refinement), the in vitro studies are a valuable and multi-faceted source of information and may explain the adequacy of further in vivo research.
Supporting information S1