1. Introduction

Cancer is one of the major world’s health problems and challenge, and its incidence and mortality are still rapidly growing worldwide, despite enormous efforts to search for cure.

Colorectal cancer (CRC) is the third leading cause of cancer death in the world, and its incidence is steadily rising in developing nations ^[1]. CRC is the second most common cause of cancer death in the United States, and the third most common cause of cancer death in both men and women in the United States ^[2][3]. However, more than one-half of cancer cases and deaths are potentially preventable due to modifiable risk factors, such as smoking, unhealthy diet, consumption of alcohol, physical inactivity and overweight and obesity ^[4]. A simple modification of diet by increasing vegetable and fruit intake, by maintenance of optimum body weight and regular physical activity, it has been shown that 30–40% of cancers could be prevented ^[5][6]. The role of natural dietary agents in cancer prevention and treatment of both solid and hematologic malignancies has been recognized as a promising tool to control the onset and progression of malignancies, because of the little of no toxicity, high efficacy in multiple sites, oral consumption, known mechanisms of action, low cost and the human acceptance ^[7][8][9]. Since diet has an important role in the etiology of CRC ^[9], that is a very heterogenous disease caused by the interaction of genetic (5–10%) and environmental factors, the use of natural products for prevention and management of CRC is extremely promising.

Colorectal cancer (CRC) is the third leading cause of cancer death in the world, and its incidence is steadily rising in developing nations [1]. CRC is the second most common cause of cancer death in the United States, and the third most common cause of cancer death in both men and women in the United States [2,3]. However, more than one-half of cancer cases and deaths are potentially preventable due to modifiable risk factors, such as smoking, unhealthy diet, consumption of alcohol, physical inactivity and overweight and obesity [4]. A simple modification of diet by increasing vegetable and fruit intake, by maintenance of optimum body weight and regular physical activity, it has been shown that 30–40% of cancers could be prevented [5,6]. The role of natural dietary agents in cancer prevention and treatment of both solid and hematologic malignancies has been recognized as a promising tool to control the onset and progression of malignancies, because of the little of no toxicity, high efficacy in multiple sites, oral consumption, known mechanisms of action, low cost and the human acceptance [7,8,9]. Since diet has an important role in the etiology of CRC [9], that is a very heterogenous disease caused by the interaction of genetic (5–10%) and environmental factors, the use of natural products for prevention and management of CRC is extremely promising.

IP6 (

myo

-inositol hexaphosphate, InsP

₆

, phytic acid) and its parent compound 

myo-inositol (Ins) are abundant in plants, particularly in cereals and legumes ^[10][11][12]. The phosphate grouping in positions 1, 2 and 3 (axial-equatorial-axial) is exclusive for IP6, providing a specific interaction with iron to completely inhibit its ability to catalyze hydroxyl radical formation, making IP6 a strong antioxidant ^[13]. However, almost all mammalian cells contain IP6 and lower inositol phosphates, although in much smaller amounts, where they are important in regulating vital cellular functions and activities, such as cell division, cellular differentiation, ion channel permeability, embryonic development, but the most important role of these versatile inositol phosphates is their role in cellular transduction systems ^{[14][15][16][17][18]}. And this unique molecule, IP6, appears to be coming from the soil to our cells via the cereal grains, a staple for much of the world population.

-inositol (Ins) are abundant in plants, particularly in cereals and legumes [10,11,12]. The phosphate grouping in positions 1, 2 and 3 (axial-equatorial-axial) is exclusive for IP6, providing a specific interaction with iron to completely inhibit its ability to catalyze hydroxyl radical formation, making IP6 a strong antioxidant [13]. However, almost all mammalian cells contain IP6 and lower inositol phosphates, although in much smaller amounts, where they are important in regulating vital cellular functions and activities, such as cell division, cellular differentiation, ion channel permeability, embryonic development, but the most important role of these versatile inositol phosphates is their role in cellular transduction systems [14,15,16,17,18]. And this unique molecule, IP6, appears to be coming from the soil to our cells via the cereal grains, a staple for much of the world population.

2. Anticancer Activity of IP6

2.1. Hypotheses and First Experiments

In testing the hypothesis that the anticancer action of IP6 may be mediated via lower phosphorylated forms of inositol, we performed experiments and showed that IP6 was not only absorbed from the gastrointestinal tract, but also taken up by malignant cells [22]. In fact, orally administered IP6 was able to reach target tumor tissue distant from the gastrointestinal tract [23]. Exogenous IP6 was rapidly taken up by mechanisms involving pinocytosis or receptor-mediated endocytosis, transported intracellularly, and dephosphorylated into inositol phosphates with fewer phosphate groups [22]. To confirm the hypothesis and the proposed mechanism of action, we followed the fate of radiolabeled IP6. Analyzing absorption, intracellular distribution, and metabolism of [

³

H]-IP6 in tumor cells by differential centrifugation and anion-exchange chromatography, we found that the predominant metabolite of IP6 was inositol tetraphosphate (IP4), indicating the possible important role of this lower IP6 metabolite in its anticancer activity [22]. Utilizing the same approach, and analyzing the uptake, distribution, and metabolism of [

³H]-IP6 in tissues, plasma, urine and feces, after it was given orally to rats bearing mammary tumors, we found that it was quickly absorbed from the gastrointestinal tract and widely distributed through the body, with a substantial amount of radioactivity found in tumor tissues ^[23]. These data showing a rapid distribution and metabolism of exogenously given IP6, at least in part explained the anticancer activity of IP6 at sites distant from the gastrointestinal tract. However, determination of non-radiolabeled IP6 in biological matrices, fluids and tissues has always been a big problem, obstacle, and challenge. Appropriate analytical methodologies were developed and utilized by Prof. Grases and his group in Spain ^[24][25]. Using inductively coupled plasma atomic emission spectrometry (ICP-AES), and later liquid chromatography-mass spectrometry (LC-MS) bioanalytical methods, they were able to develop direct, sensitive, and selective bioanalytical methods for determination of IP6 and other inositol phosphates in mammalian cells and tissues. A recently introduced the polyacrylamide gel electrophoresis (PAGE)-based method is very suitable for detection of IP6 and other inositol phosphates not only in plants, but also in animal and human cells and tissues ^[26]. Ferry et al. ^[27] added further confirmation to these data and the original hypothesis, as well as the pioneering observation that the externally applied IP6 enters the cell followed by dephosphorylation. Eiseman et al. ^[28] also observed an increase in dephosphorylated metabolites of IP6 tumor xenografts in IP6-fed mice. Furthermore, that highly negatively charged IP6 molecule is internalized by endocytosis as a cation salt and revealed that, subsequently, IP6 is completely dephosphorylated to inositol in lysosomes ^[29]. Thus, not only one of the key misconceptions about this molecule is nullified, but it has been demonstrated that IP6 is an essential nutrient whose level in plasma and urine fluctuates following deficiency or replenishment ^[24][25]. In essence, it has all the characteristics of a vitamin. Interestingly, very recently, while critically discussing employing IP6 as a new options of cancer treatment, a group from Germany postulated and confirmed almost the same hypothesis ^[30].

H]-IP6 in tissues, plasma, urine and feces, after it was given orally to rats bearing mammary tumors, we found that it was quickly absorbed from the gastrointestinal tract and widely distributed through the body, with a substantial amount of radioactivity found in tumor tissues [23]. These data showing a rapid distribution and metabolism of exogenously given IP6, at least in part explained the anticancer activity of IP6 at sites distant from the gastrointestinal tract. However, determination of non-radiolabeled IP6 in biological matrices, fluids and tissues has always been a big problem, obstacle, and challenge. Appropriate analytical methodologies were developed and utilized by Prof. Grases and his group in Spain [24,25]. Using inductively coupled plasma atomic emission spectrometry (ICP-AES), and later liquid chromatography-mass spectrometry (LC-MS) bioanalytical methods, they were able to develop direct, sensitive, and selective bioanalytical methods for determination of IP6 and other inositol phosphates in mammalian cells and tissues. A recently introduced the polyacrylamide gel electrophoresis (PAGE)-based method is very suitable for detection of IP6 and other inositol phosphates not only in plants, but also in animal and human cells and tissues [26]. Ferry et al. [27] added further confirmation to these data and the original hypothesis, as well as the pioneering observation that the externally applied IP6 enters the cell followed by dephosphorylation. Eiseman et al. [28] also observed an increase in dephosphorylated metabolites of IP6 tumor xenografts in IP6-fed mice. Furthermore, that highly negatively charged IP6 molecule is internalized by endocytosis as a cation salt and revealed that, subsequently, IP6 is completely dephosphorylated to inositol in lysosomes [29]. Thus, not only one of the key misconceptions about this molecule is nullified, but it has been demonstrated that IP6 is an essential nutrient whose level in plasma and urine fluctuates following deficiency or replenishment [24,25]. In essence, it has all the characteristics of a vitamin. Interestingly, very recently, while critically discussing employing IP6 as a new options of cancer treatment, a group from Germany postulated and confirmed almost the same hypothesis [30].

2.2. IP6 Is a Broad-Spectrum Anticancer Agent

The vast amount of laboratory data accumulated over 30 years in vitro and in vivo has confirmed the original Hypothesis I, that IP6 is indeed a broad-spectrum antineoplastic agent, effective against cancers of different cells and different tissue systems.

The most important hallmark of cancer is a sustained growth and a sustained chronic proliferation. A consistent growth inhibition of many different cancer cells has been shown for IP6 in a time- and dose-dependent fashion, and in concentrations from 0.5–5.0 mM. Antiproliferative effects of exogenous and extracellular IP6 were observed on human leukemia cells ^[31][32], human colon cancer cells ^[33] human breast cancer cell line, both estrogen receptor-positive and estrogen receptor-negative ^[34], cervical cancer ^[27], prostate cancer ^[35][36], and hepatoma cell lines ^[37]. In addition to all these epithelial cells, IP6 was able to inhibit the growth of mesenchymal tumors, such as murine fibrosarcoma ^[38] and human rhabdomyosarcoma ^[39]. However, different sensitivity to IP6 were observed with different cell types and in different models; leukemia cells and hepatocellular carcinoma cells responded more sensitive to IP6 treatment, indicating that this growth inhibitory effect was attributed to different mechanisms ^[40].

The most important hallmark of cancer is a sustained growth and a sustained chronic proliferation. A consistent growth inhibition of many different cancer cells has been shown for IP6 in a time- and dose-dependent fashion, and in concentrations from 0.5–5.0 mM. Antiproliferative effects of exogenous and extracellular IP6 were observed on human leukemia cells [31,32], human colon cancer cells [33] human breast cancer cell line, both estrogen receptor-positive and estrogen receptor-negative [34], cervical cancer [27], prostate cancer [35,36], and hepatoma cell lines [37]. In addition to all these epithelial cells, IP6 was able to inhibit the growth of mesenchymal tumors, such as murine fibrosarcoma [38] and human rhabdomyosarcoma [39]. However, different sensitivity to IP6 were observed with different cell types and in different models; leukemia cells and hepatocellular carcinoma cells responded more sensitive to IP6 treatment, indicating that this growth inhibitory effect was attributed to different mechanisms [40].

Along with this reduction in cell proliferation, rather normalization, IP6 induces differentiation and maturation of malignant cells, often resulting in reversion to the normal phenotype, as demonstrated in K-562 hematopoietic cells ^[31], human colon carcinoma HT-29 cells ^[33][41], prostate cancer cells ^[35], breast cancer cells ^[34] and rhabdomyosarcoma cells ^[39]. It was also observed that IP6 treatment of human hepatoma cell line caused a dramatic reduction in secretion of the tumor marker α-fetoprotein (AFP) ^[37]. A practical application of this effect in the clinical setting is to monitor patients with cancer for reduction of tumor markers by laboratory tests, and there have been emerging evidence of that happening clinically.

Along with this reduction in cell proliferation, rather normalization, IP6 induces differentiation and maturation of malignant cells, often resulting in reversion to the normal phenotype, as demonstrated in K-562 hematopoietic cells [31], human colon carcinoma HT-29 cells [33,41], prostate cancer cells [35], breast cancer cells [34] and rhabdomyosarcoma cells [39]. It was also observed that IP6 treatment of human hepatoma cell line caused a dramatic reduction in secretion of the tumor marker α-fetoprotein (AFP) [37]. A practical application of this effect in the clinical setting is to monitor patients with cancer for reduction of tumor markers by laboratory tests, and there have been emerging evidence of that happening clinically.

The cancer preventive activity of IP6 in vitro was tested in a benzo[

a]pyrene-induced transformation of rat tracheal epithelial cells ^[42], and then in a model using BALB/c mouse 3T3 fibroblasts ^[43] with modest efficacy. Further, IP6 was shown to be effective in prevention of skin cancer, utilizing in vitro model of skin carcinogenesis in JB6 cells ^[44] and in vivo model, where IP6 inhibited 7,12-dimethylbenz[

]pyrene-induced transformation of rat tracheal epithelial cells [42], and then in a model using BALB/c mouse 3T3 fibroblasts [43] with modest efficacy. Further, IP6 was shown to be effective in prevention of skin cancer, utilizing in vitro model of skin carcinogenesis in JB6 cells [44] and in vivo model, where IP6 inhibited 7,12-dimethylbenz[

a

]anthracene (DMBA)-induced mouse skin tumor development, indicating its potential for the management of skin tumorigenesis [45].

As stated before, the very first experiments with IP6 were investigating the effectiveness of IP6 as a cancer preventive agent in colon cancer and showed a great potential in models where cancer was induced in different species (rats and mice) and even with different carcinogens (1,2-dimethylhydrazine and azoxymethane) ^{[19][20][21][46][47][48][49][50][51]}. It is important to highlight that IP6 was effective in preventing tumor formation either given before or after carcinogen administration. However, extremely significant was a finding that IP6 was potent in reducing development of large intestinal cancer, even when treatment was begun 5 months after carcinogen initiation. Compared to untreated rats, animals on IP6 had 27% fewer tumors. These findings pointed towards its possible therapeutic use ^[21]. Aberrant crypts, putative preneoplastic lesions, have been proposed as intermediate biomarkers for colon cancer, and in these first studies of colon cancer, IP6 was shown to be effective in decreasing the incident of aberrant crypt foci in IP6-treated rats ^[48][49]. Studies were expanded to other experimental models. A consistent, reproducible, and significant inhibition of mammary cancer by IP6 was shown in experimental models chemically induced either by DMBA ^{[52][53][54][55]} or 

As stated before, the very first experiments with IP6 were investigating the effectiveness of IP6 as a cancer preventive agent in colon cancer and showed a great potential in models where cancer was induced in different species (rats and mice) and even with different carcinogens (1,2-dimethylhydrazine and azoxymethane) [19,20,21,46,47,48,49,50,51]. It is important to highlight that IP6 was effective in preventing tumor formation either given before or after carcinogen administration. However, extremely significant was a finding that IP6 was potent in reducing development of large intestinal cancer, even when treatment was begun 5 months after carcinogen initiation. Compared to untreated rats, animals on IP6 had 27% fewer tumors. These findings pointed towards its possible therapeutic use [21]. Aberrant crypts, putative preneoplastic lesions, have been proposed as intermediate biomarkers for colon cancer, and in these first studies of colon cancer, IP6 was shown to be effective in decreasing the incident of aberrant crypt foci in IP6-treated rats [48,49]. Studies were expanded to other experimental models. A consistent, reproducible, and significant inhibition of mammary cancer by IP6 was shown in experimental models chemically induced either by DMBA [52,53,54,55] or 

N-methylnitrosourea (NMU) ^[47] by us, and others, and the effect was seen on tumor incidence, tumor size, and tumor multiplicity. Anticancer effect of IP6 was also shown in murine transplanted and metastatic fibrosarcoma ^[38] and in various models of skin cancer ^{[45][56][57][58]}. Protective effect of IP6 against ultraviolet B (UVB) light, known as a complete carcinogen, was shown in SKH1 hairless mice ^[57][58].

-methylnitrosourea (NMU) [47] by us, and others, and the effect was seen on tumor incidence, tumor size, and tumor multiplicity. Anticancer effect of IP6 was also shown in murine transplanted and metastatic fibrosarcoma [38] and in various models of skin cancer [45,56,57,58]. Protective effect of IP6 against ultraviolet B (UVB) light, known as a complete carcinogen, was shown in SKH1 hairless mice [57,58].

The therapeutic properties of IP6 were first shown in the FSA-1 mouse model of transplantable and metastatic fibrosarcoma, where after subcutaneous inoculation of mouse fibrosarcoma FSA-1 cells, mice were treated with intraperitoneal injections of IP6 ^[38]. IP6 treatment resulted in increased survival and a significant inhibition of tumor size. The same model was used to monitor the development of lung metastasis after the intravenous injections of FSA-1 cells, and IP6 treatment significantly reduced the development of lung metastases. In two other models, we monitored the effects of IP6 on tumor formation, and then on tumor regression. Effect of IP6 on tumor formation was tested in human rhabdomyosarcoma RD cells transplanted in nude mice. The peritumoral treatment with IP6 (40 mg/kg) started two days after subcutaneous injection of rhabdomyosarcoma cells, three times weekly for 5 weeks. There was a 49-fold reduction of tumor size in IP6-treated animals ^[39]. To test the efficacy of IP6 on tumor regression, we used similar model for liver cancer, trying to see if IP6 would be potent in inhibiting experimental hepatoma ^[37][59]. First, a single treatment of HepG2 cells in vitro by IP6 resulted in the complete loss of the ability of these cells to form tumors when inoculated subcutaneously in nude mice ^[59]. Additionally, we demonstrated that the preexisting liver cancers regressed when treated directly with IP6 ^[59]. The treatment with intra-tumoral injection of IP6 started when tumor reached 1 cm in diameter, and after 12 days of treatment, tumor weight in IP6-treated mice was 3.4-fold less than in control mice ^[59]. Also, IP6 administered in drinking water was a potent inhibitor of in vivo growth of human prostate cancer xenografts in nude mice ^[60].

The therapeutic properties of IP6 were first shown in the FSA-1 mouse model of transplantable and metastatic fibrosarcoma, where after subcutaneous inoculation of mouse fibrosarcoma FSA-1 cells, mice were treated with intraperitoneal injections of IP6 [38]. IP6 treatment resulted in increased survival and a significant inhibition of tumor size. The same model was used to monitor the development of lung metastasis after the intravenous injections of FSA-1 cells, and IP6 treatment significantly reduced the development of lung metastases. In two other models, we monitored the effects of IP6 on tumor formation, and then on tumor regression. Effect of IP6 on tumor formation was tested in human rhabdomyosarcoma RD cells transplanted in nude mice. The peritumoral treatment with IP6 (40 mg/kg) started two days after subcutaneous injection of rhabdomyosarcoma cells, three times weekly for 5 weeks. There was a 49-fold reduction of tumor size in IP6-treated animals [39]. To test the efficacy of IP6 on tumor regression, we used similar model for liver cancer, trying to see if IP6 would be potent in inhibiting experimental hepatoma [37,59]. First, a single treatment of HepG2 cells in vitro by IP6 resulted in the complete loss of the ability of these cells to form tumors when inoculated subcutaneously in nude mice [59]. Additionally, we demonstrated that the preexisting liver cancers regressed when treated directly with IP6 [59]. The treatment with intra-tumoral injection of IP6 started when tumor reached 1 cm in diameter, and after 12 days of treatment, tumor weight in IP6-treated mice was 3.4-fold less than in control mice [59]. Also, IP6 administered in drinking water was a potent inhibitor of in vivo growth of human prostate cancer xenografts in nude mice [60].

2.3. Mechanism of Action and Molecular Interactions of IP6

Though we know that the cellular mechanisms involved in the anticancer activity of IP6 and Ins involve cell proliferation and differentiation, we now better understand the involvement of these molecules in biochemical pathways and their molecular interaction. It is known that virtually all animal cells contain inositol phosphates that have important role in cellular signal transduction, regulation of cell function, growth, and differentiation ^{[14][15][16][17][18]}. The role of IP6 among multiple signaling pathways and their cross-talks in regulation of cell function is complex and still needs to be addressed in the future.

Though we know that the cellular mechanisms involved in the anticancer activity of IP6 and Ins involve cell proliferation and differentiation, we now better understand the involvement of these molecules in biochemical pathways and their molecular interaction. It is known that virtually all animal cells contain inositol phosphates that have important role in cellular signal transduction, regulation of cell function, growth, and differentiation [14,15,16,17,18]. The role of IP6 among multiple signaling pathways and their cross-talks in regulation of cell function is complex and still needs to be addressed in the future.

Detailed and elaborate reviews of the anticancer activity of IP6 and Ins have been published ^{[30][40][61][62][63]}. Here we highlight some of the most important cellular mechanisms and molecular targets of IP6 and focus on those important in colon cancer research.

Detailed and elaborate reviews of the anticancer activity of IP6 and Ins have been published [30,40,67,68,69]. Here we highlight some of the most important cellular mechanisms and molecular targets of IP6 and focus on those important in colon cancer research.

IP6 can modulate cellular response at the level of receptor binding; after sterically blocking the heparin-binding domain of basic fibroblast growth factor, IP6 disrupted further receptor interactions ^[64]. In addition to acting on phosphatidylinositol-3 kinase and activating protein-1 by IP6 ^[44], protein kinase C ^[65][66] and mitogen-activated protein kinases ^[44][67] are involved in IP6-mediated anticancer activity. Various cellular proteins, pathways and mechanisms are involved in the observed anticancer effects of IP6. In addition to the growth inhibition, induction of differentiation and apoptosis, inhibition of angiogenesis and suppression of tumor metastasis are observed ^{[30][40][61][62][63]}. Critical molecular targets are involved in these cellular processes and activities. IP6 downregulates p21 and p27, inhibits pRB phosphorylation and cell cycle progression ^[66], targets PI3K/Akt pathway and counteracts the activation of PKC/RAS/ERK pathway ^{[30][61][62][66]}. Furthermore, by downregulating Akt and ERK, IP6 reduces NF-kappaB and inhibits inflammation ^[30][61][62]. In summary, IP6 impacts various intracellular signaling pathways and networks, and expression of genes encoding key cellular proteins, such as p53, p21, p27, BCL-2, and MMPs. It seems that the inhibition of the phosphorylation-based activation of key molecular targets is a basic mechanism through which IP6 interferes with specific cellular and biological functions ^[61]. Very recently, the regulation of Cullin-RING E3 ligase dynamics by IP6 has been suggested ^[68]. Most of the current therapeutic molecular glues that target disease-causing proteins for degradation utilize the largest family of ubiquitin ligases in humans, the Cullin-RING (Really Interesting New Gene) ligases (CRLs). Because, IP6 causes G1 arrest by down-regulating p21 and p27, and since both p21 and p27 are well-defined substrates for CRL1- and CRL4-based ubiquitin ligases, it has been tempting to speculate that at least some degree of IP6’s anticancer activity might come from increased down-regulation of CRL function, thus stabilizing p21 and p27 to promote G1 arrest ^[68].

IP6 can modulate cellular response at the level of receptor binding; after sterically blocking the heparin-binding domain of basic fibroblast growth factor, IP6 disrupted further receptor interactions [70]. In addition to acting on phosphatidylinositol-3 kinase and activating protein-1 by IP6 [44], protein kinase C [71,72] and mitogen-activated protein kinases [44,73] are involved in IP6-mediated anticancer activity. Various cellular proteins, pathways and mechanisms are involved in the observed anticancer effects of IP6. In addition to the growth inhibition, induction of differentiation and apoptosis, inhibition of angiogenesis and suppression of tumor metastasis are observed [30,40,67,68,69]. Critical molecular targets are involved in these cellular processes and activities. IP6 downregulates p21 and p27, inhibits pRB phosphorylation and cell cycle progression [72], targets PI3K/Akt pathway and counteracts the activation of PKC/RAS/ERK pathway [30,67,68,72]. Furthermore, by downregulating Akt and ERK, IP6 reduces NF-kappaB and inhibits inflammation [30,67,68]. In summary, IP6 impacts various intracellular signaling pathways and networks, and expression of genes encoding key cellular proteins, such as p53, p21, p27, BCL-2, and MMPs. It seems that the inhibition of the phosphorylation-based activation of key molecular targets is a basic mechanism through which IP6 interferes with specific cellular and biological functions [67]. Very recently, the regulation of Cullin-RING E3 ligase dynamics by IP6 has been suggested [74]. Most of the current therapeutic molecular glues that target disease-causing proteins for degradation utilize the largest family of ubiquitin ligases in humans, the Cullin-RING (Really Interesting New Gene) ligases (CRLs). Because, IP6 causes G1 arrest by down-regulating p21 and p27, and since both p21 and p27 are well-defined substrates for CRL1- and CRL4-based ubiquitin ligases, it has been tempting to speculate that at least some degree of IP6’s anticancer activity might come from increased down-regulation of CRL function, thus stabilizing p21 and p27 to promote G1 arrest [74].

Investigating the anticancer effect of IP6 in colon cancer, the involvement of already mentioned intracellular signaling pathways and critical targets have been shown in a broad-spectrum activities from the effect on aberrant crypt foci, an early biomarker of colon carcinogenesis ^[30][40] to the regulation of microRNA-155 and its related genes expression ^[69]. Exploring the effect of combined IP6 and Ins on the appearance of colorectal cancer metastasis to the liver, Dr. Song and his group conducted several studies. Here, utilizing a splenic capsule, first they were trying to see the effect, and after to explore and understand the mechanism of this metastatic spread ^[70]. Tumor metastasis is a complex process, involving adhesion, migration, intra- and extravasation, colonization, and proliferation, with alterations of multiple mechanisms. Major components of the cell-extracellular interactions are collagen IV, fibronectin, and laminin, and IP6 and Ins treatment altered the expression of all these proteins. Additionally, MMP-9, a type IV collagenase, and its high expression is closely correlated with malignant tumor invasion, metastasis and vascular formation, and IP6 and Ins were shown to inhibit the MMP-9 in this model ^[70]. Very recently, in the follow-up study, the same group investigated the liver metastasis of colorectal cancer, this time using an orthotopic transplantation model of colorectal cancer to monitor the effect of IP6, Ins and their combination. First, they show again, that the IP6 + Ins was more effective in inhibiting the liver metastasis of colon cancer than each compound administered individually. Additionally, regulation of mutation of Wnt/β-catenin signaling pathway by inhibiting Wnt10b, Tcf7, β-catenin, and c-Myc from abnormally high expression is critical to increase efficacy ^[71].

Investigating the anticancer effect of IP6 in colon cancer, the involvement of already mentioned intracellular signaling pathways and critical targets have been shown in a broad-spectrum activities from the effect on aberrant crypt foci, an early biomarker of colon carcinogenesis [30,40] to the regulation of microRNA-155 and its related genes expression [75]. Exploring the effect of combined IP6 and Ins on the appearance of colorectal cancer metastasis to the liver, Dr. Song and his group conducted several studies. Here, utilizing a splenic capsule, first they were trying to see the effect, and after to explore and understand the mechanism of this metastatic spread [64]. Tumor metastasis is a complex process, involving adhesion, migration, intra- and extravasation, colonization, and proliferation, with alterations of multiple mechanisms. Major components of the cell-extracellular interactions are collagen IV, fibronectin, and laminin, and IP6 and Ins treatment altered the expression of all these proteins. Additionally, MMP-9, a type IV collagenase, and its high expression is closely correlated with malignant tumor invasion, metastasis and vascular formation, and IP6 and Ins were shown to inhibit the MMP-9 in this model [64]. Very recently, in the follow-up study, the same group investigated the liver metastasis of colorectal cancer, this time using an orthotopic transplantation model of colorectal cancer to monitor the effect of IP6, Ins and their combination. First, they show again, that the IP6 + Ins was more effective in inhibiting the liver metastasis of colon cancer than each compound administered individually. Additionally, regulation of mutation of Wnt/β-catenin signaling pathway by inhibiting Wnt10b, Tcf7, β-catenin, and c-Myc from abnormally high expression is critical to increase efficacy [65].

2.4. Sensitivity and Selectivity of IP6

Almost all the anticancer agents have severe side effects on normal tissues and organs. IP6 was shown that while it can sensitize cancer cells for treatment, at the same time can offer a protection to normal tissues. This sensitivity and selectivity of IP6 is its unique property. In one of the first studies in Italy, using bone marrow and isolated fresh CD34

⁺ cells from bone marrow and observing the treatment with different dose of IP6. While IP6 was very effective against the leukemic progenitors from chronic myelogenous leukemia patients, no cytotoxic or cytostatic effect was observed on normal bone marrow progenitor cells under the same conditions ^[32]. Investigating the effect of IP6 on Kaposi Sarcoma, IP6 inhibited the colony formation of Kaposi Sarcoma cell lines, KS Y-1 (AIDS-related KS cell line) and KS SLK (Iatrogenic KS), and CCRF-CEM (human adult T lymphoma) cells in a dose-dependent manner ^[72]. However, IP6 did not affect the ability of normal cells (peripheral blood mononuclear cells and T cell colony-forming cells) to form colonies in a semisolid methylcellulose medium ^[72]. The activity of IP6 was compared to taxol, that was used as a control, and that contrary to IP6 affected the colony formation of normal cells. A similar effect was observed when the response of MCF7-10A cells, a normal human breast epithelial cells was compared with the breast cancer cell lines MCF-7 and MDA-MB-231. While the growth of the epithelial cells was not affected, the proliferation of cancer cells was significantly reduced after the incubation with IP6 due to the increased rate of apoptosis ^[73]. There are known differences between malignant and normal cells in their behavior, known as the hallmarks of cancer, and described by Hannahan and Weinberg, and differences in metabolism, known as Warburg effect. However, this selectivity of IP6 for normal and malignant cells deserves further investigation.Furthermore, IP6 was shown to acts synergistically with standard chemotherapeutics. Cancer therapy recognizes the importance of using combination therapy with rationale to increase efficacy and decrease side-effects of conventional chemotherapy. Another important aspect of combination therapy is overcoming acquired drug resistance. It was demonstrated that IP6 acted synergistically with doxorubicin and tamoxifen, being particularly effective against estrogen receptor-negative and doxorubicin-resistant cell lines, both conditions that are very challenging to treat ^[74]. These data are particularly important because tamoxifen is usually given as a chemopreventive agent in the post-treatment period, but also because doxorubicin has enormous cardiotoxicity and its use is associated with doxorubicin resistance, both conditions improved by IP6.

 cells from bone marrow and observing the treatment with different dose of IP6. While IP6 was very effective against the leukemic progenitors from chronic myelogenous leukemia patients, no cytotoxic or cytostatic effect was observed on normal bone marrow progenitor cells under the same conditions [32]. Investigating the effect of IP6 on Kaposi Sarcoma, IP6 inhibited the colony formation of Kaposi Sarcoma cell lines, KS Y-1 (AIDS-related KS cell line) and KS SLK (Iatrogenic KS), and CCRF-CEM (human adult T lymphoma) cells in a dose-dependent manner [78]. However, IP6 did not affect the ability of normal cells (peripheral blood mononuclear cells and T cell colony-forming cells) to form colonies in a semisolid methylcellulose medium [78]. The activity of IP6 was compared to taxol, that was used as a control, and that contrary to IP6 affected the colony formation of normal cells. A similar effect was observed when the response of MCF7-10A cells, a normal human breast epithelial cells was compared with the breast cancer cell lines MCF-7 and MDA-MB-231. While the growth of the epithelial cells was not affected, the proliferation of cancer cells was significantly reduced after the incubation with IP6 due to the increased rate of apoptosis [79]. There are known differences between malignant and normal cells in their behavior, known as the hallmarks of cancer, and described by Hannahan and Weinberg, and differences in metabolism, known as Warburg effect. However, this selectivity of IP6 for normal and malignant cells deserves further investigation.Furthermore, IP6 was shown to acts synergistically with standard chemotherapeutics. Cancer therapy recognizes the importance of using combination therapy with rationale to increase efficacy and decrease side-effects of conventional chemotherapy. Another important aspect of combination therapy is overcoming acquired drug resistance. It was demonstrated that IP6 acted synergistically with doxorubicin and tamoxifen, being particularly effective against estrogen receptor-negative and doxorubicin-resistant cell lines, both conditions that are very challenging to treat [80]. These data are particularly important because tamoxifen is usually given as a chemopreventive agent in the post-treatment period, but also because doxorubicin has enormous cardiotoxicity and its use is associated with doxorubicin resistance, both conditions improved by IP6.