Inhibitory Effects of Vitamin A and Its Derivatives on Cancer Cell Growth Not Mediated by Retinoic Acid Receptors

Abstract

Vitamin A is an important trace essential nutrient. Vitamin A is present as a retinyl ester in animal foods and as β-carotene (provitamin A), which is a precursor of vitamin A, in plant foods such as green and yellow vegetables. After ingestion and absorption in the body, these are converted into retinol and stored as retinyl esters in stellate cells in the liver. The stored retinyl esters are decomposed into retinol as needed, and converted into the aldehyde retinal, which plays an important role in vision. Retinoic acid (RA) has a variety of effects. In particular, RA is used as a therapeutic agent for acute promyelocytic leukemia. This review will cover (1) elucidation of anti-refractory cancer effects of retinol (vitamin A) not mediated by RA receptors, (2) elucidation of anti-cancer effects of RA not mediated by RA receptors and (3) the development of candidate new anti-cancer agents that combine the actions of RA and retinol. Lessons learned from these findings are that vitamin A has anti-cancer activity not mediated by RA receptors; that nutritional management of vitamin A leads to prevention and treatment of cancer, and that new compounds developed from RA derivatives represent good anti-cancer drug candidates that are in various stages of clinical trials.

1. INTRODUCTION

Vitamin A was discovered in 1913, approximately 109 years ago.¹⁾ Although various physiological effects of vitamin A have been identified, not all functions of vitamin A have yet been elucidated.²⁾ Vitamin A is an essential nutrient. It is a general term for various related compounds. The main compounds of vitamin A are provitamin A, such as β-carotene, which is contained in plant foods; retinyl ester and retinol, both of which are contained in animal foods and metabolites, such as retinal and retinoic acid (RA) (Fig. 1A). β-Carotene is ingested and absorbed and then decomposed into the aldehyde retinal. This can be further reduced to retinol, and then stored in the stellate cells of the liver in the form of retinyl esters (Fig. 1B). The stored retinyl esters can hydrolyzed to retinol and oxidized into retinal and RA to exert various actions. Most of the vitamin A in the circulatory system is retinol. Physiological actions of vitamin A include the effects of retinal³⁾; growth and differentiation; formation of mucous membranes of the skin; immune-stimulatory functions, and anticancer actions, etc. These are known and play a wide range of important roles.^4,5) It is thought that these effects are not due to retinol, but rather to RA.

Fig. 1. (A) Vitamin A and (B) Its Biocirculation, and Physiological Actions

Cancer has been the leading cause of death for Japanese in Japan since 1981, with more than 300000 people dying from cancer every year. For cancer treatment, surgical resection of affected tissues and drug treatment with anticancer drugs are frequently employed. Anti-cancer drugs are compounds with a variety of medicinal properties. Although they can be quite effective, they are not yet sufficient and have problems. There is a need to develop effective drugs for insensitive cancers, such as drug-resistant cancers and refractory cancers, which have low 5-year survival rates. There is also a need to develop anticancer drugs with low side effects. There is a relationship between vitamin A and cancer, and RA has already been used to treat acute promyelocytic leukemia (APL).

The discovery of the differentiation-inducing effects of RA on the human promyelocytic leukemia cell line HL60 confirmed the effect of suppressing the growth of cancer cells.⁶⁾ Next, the mechanism of action of RA was investigated. This clarified that nuclear receptors bind to RA, which recognize and bind to a specific base sequence (RARE) of DNA to control the expression of downstream genes, and in so doing, result in RA actions.^7,8) RA is used as a therapeutic drug for the treatment of APL based on its cell differentiation-inducing effects. However, relapsed patients become RA-resistant and RA becomes ineffective.^9–11) It is also known that RA does not suppress the growth of insensitive cancers (refractory/drug-resistant cancers).^12–15)

As described above, research on vitamin A and cancer is predominantly based on the premise of RA nuclear receptor-mediated action. However, there are few studies of retinol, which has a biological content 100 times higher than that of RA. Therefore, in our research on the effects of retinol on cancer cells, we found (1) that the anticancer effects of retinol are not mediated by RA receptors. From there, (2) we discovered that RA has an anticancer effect that is not mediated by RA receptors. Furthermore, based on research results with retinol and RA, (3) we succeeded in developing a new anticancer drug. We will explain these three achievements in turn.

2. ELUCIDATION OF ANTI-REFRACTORY CANCER ACTION OF RETINOL (VITAMIN A) NOT MEDIATED BY RA RECEPTOR (1)

Very few studies have focused on retinol. Actions of retinol has long been thought to be due to RA. The possibility that retinol has its own physiological function has been largely overlooked. For example, the immune system deficiency of vitamin A-deficient rodents is not cured by RA alone.¹⁶⁾ In addition, spermatogenesis is impaired in vitamin A deficiency, but RA administration does not cure this.^17–22) These hint at another role unique to the retinol. However, the mechanism was unclear.

2.1. Effects of Retinol on the Growth of Refractory Human Cancer Cells

First, using 4 types of human refractory cancer cells, gallbladder cancer cells (NOZ C-1), cholangiocarcinoma (HuCCT1), and 2 types of pancreatic cancer cells (MIA Paca2, JHP-1), the growth-suppressing effects of retinyl palmitate (RP), RA, and retinol were investigated. As a result, retinol suppressed the growth of these four types of refractory cancer cells in vitro in a concentration-dependent manner, but RA or RP had limited effects.^13,15) The IC₅₀ values of retinol obtained from the cell proliferation suppression curve were 8 µM for MIA Paca2, 6.5 µM for JHP-1, 5 µM for HuCCT1, and 14.2 µM for NOZ C-1, RA and RP were >20 µM (Table 1). The IC₅₀ value of retinol was close to the in vivo retinol concentration.

Table 1. Inhibition of Growth and Adhesion of Refractory Cancer Cells by Vitamin A

Modified with permission from tables of Biol. Pharm. Bull., 40, 495–503 (2017) and Pharmacol. Ther., 230, 107942 (2022).

2.2. Effect of Retinol on Human Refractory Cancer Cell Adhesion

Next, the effects of retinol or RA treatment on the adhesion of refractory cancer cells were investigated. Adhesion of four refractory cancer cells was inhibited in vitro by retinol treatment.¹³⁾ In cells other than JHP-1 cells, retinol strongly suppresses the adhesion of refractory cancer cells compared to RA (Table 1). These results suggested that retinol inhibits the growth, metastasis, and invasion of cancer cells, which are deeply involved in cancer progression, more strongly than RA.

2.3. Blood Retinol Concentration in 4 Types of Refractory Cancer Mice

The results described above suggest that retinol has a stronger inhibitory effect on the growth of refractory cancer cells in vitro than RA and RP. Therefore, four types of refractory cancer cells (NOZ C-1, HuCCT1, MIA Paca2, and JHP-1 cells) were injected subcutaneously into nude mice, and the blood retinol concentration of the cancer-bearing model mice was quantified by HPLC on the 28th day after transplantation of refractory cancer cells. The blood retinol levels of all cancer-bearing mice were significantly lower than those of non-transplanted control mice.^15,23) This indicates that the progression of cancer affects the metabolism of retinoids (vitamin A) in the living body and reduces the concentration of retinol in the blood.

2.4. Effect of Retinol on the Growth of Refractory Cancer Tumors and the Effect on Engraftment and Proliferation of Refractory Cancer Cells

From the above results, we thought that increasing blood retinol concentrations in vivo might suppress the growth of cancer. In order to investigate the effect of retinol on the growth of refractory cancer tumors and the effects on engraftment and proliferation of refractory cancer cells, the effects of administration of RP as a substitute for retinol were investigated.

2.4.1. Effect of RP on Refractory Cancer Tumor Growth

First, the effect of RP on tumor growth in refractory cancer mice was investigated. The NOZ C-1 strain of gallbladder cancer cells, which are refractory cancer cells, was transplanted into nude mice to prepare cancer-bearing mice, and 1000 or 2500 IU of RP was continuously administered for 21 d and then serum retinol concentration and tumor formation weight were measured. We found that serum retinol levels increased at both doses of RP treatment. With this, the tumor weight of refractory cancer decreased significantly.²³⁾ Therefore, RP administration increased the blood retinol concentration in refractory cancer-bearing mice and suppressed tumor growth, indicating that the two are related.

2.4.2. Effect of RP on Engraftment and Proliferation of Refractory Cancer Cells

Next, the effects of RP administration on engraftment and proliferation of refractory cancer cells transplanted into mice were investigated. Nude mice were first administered 2500 IU of RP, and 10 d later, the gallbladder cancer cell NOZ C-1 strain of refractory cancer cells was transplanted into mice, and 10 d later, serum and liver retinol concentrations and the tumor formation weight were measured. The results showed a significant increase in blood and liver retinol levels in RP-treated mice. With this, the tumor weight of refractory cancer decreased significantly.²³⁾ Therefore, increasing the blood retinol concentration suppresses the engraftment and growth of refractory cancer, suggesting that RP may be effective in cancer prevention. Furthermore, this was highly suggestive that the regulation and control of the concentration of retinol in the body could lead to the prevention and treatment of cancer.

2.5. Anti-refractory Cancer Action Mechanism of Retinol

2.5.1. Mechanism of Anticancer Action of Retinol not Mediated by RA Receptor

Previously, the action of the vitamin A or retinol (vitamin A in the narrow sense), was thought to be expressed via the RA receptor-mediated pathway. In the circulatory system, retinol bound to retinol-binding protein (RBP) is taken up into cells via STRA6 in the cell membrane that recognizes RBP, and is oxidized to retinal and then to RA, and RA is transferred to the nucleus, and regulate downstream gene expression by binding to the RA receptor. However, differences found in the following three experimental results (1.–3.) of retinol and RA (cell proliferation inhibitory effect, intracellular uptake amount, and effect of increasing the mRNA expression level of RA receptor), indicated that the mechanism of anticancer effect of retinol does not involve the RA receptor.^15,24)

1. 1.    Refractory cancer cell growth inhibitory effect: retinol > RA
   1. When the concentration dependence of retinol and RA on the growth of refractory cancer cells was investigated, retinol showed a significant concentration-dependent suppression of cancer cell growth according to the treatment. However, RA was not effective even at 20 µM treatment.

2. 2.    Intracellular uptake: retinol < RA
   1. When the time course of cellular uptake of retinol and RA was examined, the uptake of retinol reached its maximum and plateaued at approximately 30 min. After this, the amount of retinol uptake was less than half of the amount of RA uptake.

3. 3.    Effect of increasing the mRNA expression level of RA receptor: retinol < RA
   1. Retinol was lower than RA in the effect of increasing the expression of the RNA level of RA receptors (α, β, γ). These results suggest that the actions of retinol on cancer cell growth inhibitory action may arise from another mechanism not mediated by RA receptors.²⁴⁾

2.5.2. Another Mechanism of Action of Retinol Not Mediated by RA Receptor

As other mechanisms not mediated by RA receptors were indicated, apoptosis (caspase-3 activation, Bcl-2 family expression), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) were investigated. These are known as cell proliferation pathways. We found that these pathways were not involved in retinol action. Therefore, a comprehensive analysis of gene expression by microarray was done. This revealed that retinol has a significant effect on the expression of gene clusters related to endoplasmic reticulum (ER) stress.²⁴⁾

When the mRNA expression levels of various ER stress index genes by retinol were measured, the mRNA expression levels of HMOX-1 (heme oxygenase 1), which is a stress response index; CCAAT/enhancer-binding protein homologous protein (CHOP), which is an ER stress index, and 78 kDa glucose-regulated protein (GRP78), which is an ER chaperone, were significantly increased by retinol treatment. This indicated that retinol induces mRNA associated with the ER stress index (Fig. 2A). Next, when the change in the expression of the autophagy index protein by retinol treatment was examined, the protein expression level of LC3-II, which is an autophagy index, was significantly increased by retinol treatment. Therefore, retinol induces autophagy (Fig. 2B). In addition, retinol treatment significantly increased the proportion of cells in the G₀/G₁ phase of the cell cycle and significantly decreased the proportion of cells in the S phase, indicating that the cell cycle is arrested at G₀/G₁.²⁴⁾ The mechanism of expression of the anticancer effect of retinol was found to be related to ER stress and autophagy.

Fig. 2. Effects of Retinol on (A) mRNA Expression of ER Stress Markers and (B) Protein Expression of Autophagy Markers

Reproduction with permission from figure of Biol. Pharm. Bull., 40, 495–503 (2017).

2.6. Summary—Retinol

1. 1.    Retinol suppresses the growth and adhesion of refractory cancer cells in vitro and in vivo. RA has almost no such effect.
2. 2.    The blood retinol concentration of refractory cancer cell-bearing mice is lower than that of normal mice.
3. Blood retinol concentration increases and tumor formation decreases with RP administration.
4. ➔ Controlling the concentration of retinol in the body leads to prevention and treatment of cancer.
5. 3.    Retinol acts without the intervention of RA receptors. The induction of ER stress and autophagy is involved in the action mechanism of retinol.
6. 4.    The Retinol action is expressed by stopping in the cell cycle G₀/G₁.

3. ELUCIDATION OF ANTICANCER EFFECT NOT MEDIATED BY RA RECEPTOR (2)

Since we found that retinol has actions not mediated by RA receptors, we predicted that RA may also have actions not mediated by RA receptors. In our previous research, we discovered a novel retinoylation reaction, in which RA covalently binds to a protein without the intervention of the RA receptor.^15,25–31) This is in addition to RA action, in which RA is bound to a receptor with hydrogen-bonds and regulates gene expression. This retinoylation reaction proceeds via a two-step enzymatic reaction, as shown in Fig. 3.^15,32,33) The RA antibody method for detecting retinoylated proteins, described below, was an idea based on this enzymatic reaction.

Fig. 3. Retinoylation Reactions

3.1. Development and Identification of a Novel Detection Method for Retinoylated Proteins

Initially, research on protein retinoylation was slow. The reason was due to problems with the methods of detecting retinoylation, which we refer to as the “conventional method.” The conventional method utilizes autoradiography, in which cells are treated with radiolabeled RA, free RA is removed, and then autoradiography detects proteins in electrophoretically separated gels (autoradiography method). In the two-dimensional electrophoresis pattern of RA-treated HL60 cells, more than 20 proteins were found to be retinoylated.³⁴⁾ The major retinoylated protein was identified as the intermediate filament vimentin,^35,36) which plays an important role in mitosis of cells, and the sub (accessory) retinoylated protein was identified as the regulatory subunit RIIα of protein kinase A (PKA), which is important for signal transduction.^37,38) However, this conventional method required 3 to 12 months to detect the retinoylated protein, which highly problematic.

Therefore, we developed a new RA antibody methodology using RA antibodies (antibody method).³⁹⁾ With this method, retinoylated protein can be detected in 3 to 6 d. The method is to separate the total extracted protein obtained from RA-treated cells with a MonoQ column and to detect the retinoylated protein by immunostaining using RA antibody. As a result, Fig. 4A shows the protein elution pattern, and Fig. 4B shows the retinoylated protein detected by the antibody method. By this antibody methodology, all the proteins (a) PKA-RIα,³⁷⁾ (b) PKA-RIIα,³⁷⁾ and (c) Vimentin³⁶⁾ identified by the conventional method can be detected, and further, new proteins were detected; (d) Histones,⁴⁰⁾ (e) α-Actinin,³⁹⁾ and (f) Rho-GDIβ (Rho GDP-dissociation inhibitor beta).⁴¹⁾ In particular, we discovered that histones are retinoylated, because retinoylated proteins in the void fraction of the MonoQ column matched histones stained with histone antibody (Fig. 4B) and histones obtained by acid extraction from RA-treated HL60 cells were stained with RA antibody.⁴⁰⁾ In addition, we found that RA treatment changed the level of histone modification (acetylation, ubiquitination, phosphorylation, etc.), and that the significantly increased phosphorylation was due to PKA.

Fig. 4. Retinoylated Proteins Detected Using RA Antibodies in Fractions Separated by MonoQ Column Chromatography

(A) Protein elution pattern. (B) Blue: Retinoylated proteins identified by autoradiography method; Red: Retinoylated proteins identified by antibody method. Modified with permission from figures of J. Biochem., 144, 349–355 (2008) and Biol. Pharm. Bull., 32, 1943–1946 (2009).

3.2. Role of Retinoylated PKA-RIIα in Cell Differentiation and Cell Proliferation Suppression

The PKA regulatory subunit (PKA-RIIα) is retinoylated.³⁷⁾

PKA is a holoenzyme in which two regulatory subunits (R) and two catalytic subunits (C) are bound. When cAMP binds to the regulatory subunit, the dissociated catalytic subunit phosphorylates substrate proteins. In contrast, retinoylated RIIα is localized in the nucleus and it is covalently bonded to RA by an ester bond.³⁷⁾ Approximately 1.4 molecules of RA are bound to 1 molecule of RIIα, and retinoylated RIIα is detected 5 min after RA treatment.^15,42)

3.2.1. Involvement of PKA in Differentiation

In order to investigate the possibility that RA-induced retinoylated PKA is involved in the differentiation functions of HL60 cells, we examined the expression level of CD11b, which is a differentiation index. Expression of CD11b increased in RA-treated HL60 cells, but decreased with the addition of the PKA activity inhibitor Myr. PKI (PKA Inhibitor 14-22 Amide, Cell-Permeable, Myristoylated).⁴³⁾ From this result, we concluded that the expression of the differentiation index CD11b by RA treatment in HL60 cells is related to the retinoylated PKA activity.

3.2.2. Effect of RA on Protein or mRNA Levels of PKA-RIIα

Next, to clarify the roles of retinoylated PKA in cell differentiation, we analyzed changes in the intracellular dynamics of PKA due to RA. First, when protein levels were examined using immunoblotting, we found that RA treatment increased PKA-RIIα but hardly changed Cα.⁴⁴⁾ Next, when the amount of mRNA was measured using the RT-PCR method, no change was observed in the level of RIIα transcript by RA treatment. From the above results, we concluded that the increase in the amount of RIIα protein by RA treatment may be due to the stabilization of RIIα protein, and not due to the increase in mRNA expression of RIIα.

3.2.3. Stabilization of PKA-RIIα by RA Treatment

Therefore, in order to investigate the metabolism of PKA-RIIα protein in RA-treated cells, cyclohexamide, which is a protein synthesis inhibitor, was administered and the cells were recovered after 3 and 6 h, and the amount of RIIα protein was measured. In the untreated case, the amount of protein decreased over time due to metabolism, whereas with RA treatment, there was minimal effect.⁴⁴⁾ We concluded that this suggests that RA promotes stabilization by suppressing the metabolism of PKA-RIIα.

3.2.4. Changes in Cell Localization of PKA-RIIα and PKA-Cα Due to RA Treatment

Next, cell immunofluorescence staining was performed to investigate changes in the intracellular localization of RIIα and Cα due to RA treatment. RA treatment increased RIIα in the nucleus, and RA treatment also increased Cα in the nucleus.⁴⁴⁾ These results were also confirmed by cell fractionation, suggesting that RA treatment may have transferred PKA to the nucleus by holoenzyme.

3.2.5. Effects of RA on the Affinity of RIIα with A-Kinase Anchor Protein (AKAP)

In order to elucidate the mechanism of nuclear translocation of PKA after RA treatment, the effects of RA on the affinity with the AKAP that binds to importin and which plays the role of cargo for nuclear translocation, and moves to the nucleus, were examined. This showed that RA treatment increases the affinity between retinoylated RIIα and AKAP and that AKAP bound to importin may migrate to the nucleus.

3.2.6. Effects of RA on the PKA Activity

When the effect of RA on the PKA activity in the nuclear fraction was examined, the PKA activity in nucleus of RA-treated cells increased approximately 1.6 times that of untreated cells.⁴⁴⁾ Therefore, RA treatment increased the PKA phosphorylation activity in the nucleus.

3.2.7. Increase of Nuclear Proteins Phosphorylated by PKA after RA Treatment

Next, in order to detect the protein phosphorylated by PKA in the nucleus, the proteins contained in the nuclear fraction are separated by two-dimensional polyacrylamide gel electrophoresis and then immunoblotting using anti-phosphorylated-PKA substrate antibody. As a result, four PKA-phosphorylated proteins were significantly increased by RA treatment compared to vehicle (indicated by red arrows in Fig. 5A). Phosphorylation of all four proteins increased by RA treatment were suppressed by the combined use of PKA inhibitors, Rp-cAMP (Rp-Adenosine 3′,5′-cyclic monophosphorothioate triethylammonium salt hydrate) and Myr. PKI (PKA Inhibitor 14-22 Amide, Cell-Permeable, Myristoylated).⁴⁴⁾ These results revealed RA-dependent nuclear PKA activation and the concomitant presence of multiple nuclear proteins that are phosphorylated by PKA. Based on these results, we undertook a series of studies on changes in PKA due to RA treatment and these let to our conclusion that the mechanisms of cancer cell growth inhibitory action of RA are not all mediated by RA receptors (Fig. 5).

Fig. 5. Nuclear Proteins Phosphorylated by Retinoylated PKA

Modified with permission from figures of Biochim. Biophys. Acta Gen. Subj., 1861, 276–285 (2017) and Pharmacol. Ther., 230, 107942 (2022).

1. 1.    First, PKA-RIIα is retinoylated by RA treatment.
2. 2.    Retinoylation stabilizes PKA-RIIα and increases the affinity of AKAP, thereby inducing the translocation of PKA to the nucleus.
3. 3.    PKA transferred to the nucleus binds to cAMP, and the dissociated Cα phosphorylates the target nuclear proteins and suppresses proliferation.
   1. One substrate phosphorylated by nuclear PKA is the splicing factor (SF2).

3.2.8.  SF2

SF2 is a protooncogene and is involved in alternative splicing of Mcl-1 that suppresses apoptosis and vascular endothelial growth factor (VEGF), which has angiogenic effect, and suppresses exon skipping. The experimental results are as follows.⁴³⁾

1. 1.   RA treatment reduces SF2 protein level and increases SF2 phosphorylated by PKA.
2. 2.    RA treatment increases the ratio of Mcl-1S, which has an apoptotic effect, to Mcl-1L, which has an anti-apoptotic effect (Mcl-1S/Mcl-1L).
3. 3.    Cell differentiation induced by RA treatment, is suppressed in combination with PKA inhibitor and promoted in combination with Mcl-1L inhibitor.

RA treatment increases the phosphorylation of SF2 by retinoylated PKA and inactivates SF2, which promotes exon skipping, increases Mcl-1S, and inhibits the action of Mcl-1L. Finally, these lead to the induction of cell differentiation and then the suppression of cell proliferation. A PKA inhibitor suppressed the increased expression of Mcl-1S by RA and inhibited RA-induced differentiation. In contrast, Mcl-1L inhibitors promoted RA-induced differentiation. This indicated that phosphorylation by retinoylated PKA inactivates SF2, promotes exon skipping, induces differentiation, and ultimately stops proliferation.⁴³⁾

3.3. Summary—RA

As mentioned above, the following points have been clarified in the elucidation of the anticancer effect of RA not mediated by the RA receptor.

1. Development of a novel RA antibody method to detect retinoylated proteins

2. Identification of new histone and other retinoylated proteins using a novel RA antibody method

3. Analysis of the relationship between retinoylated proteins and cell proliferation inhibitory effects

Our work clarifies that retinoylation increases cell proliferation by increasing signal molecules such as PKA in the nucleus and promoting phosphorylation of nuclear proteins including histones and splicing regulators, etc. We also found that histone retinoylation affects histone modifications (acetylation and phosphorylation, etc.), Therefore, retinoylation is considered to be a reaction that changes the localization, activity, and stability of signal molecules involved in proliferation and regulates epigenetics.

4. DEVELOPMENT OF NEW ANTI-CANCER AGENTS (COMBINED RA ACTION AND RETINOL ACTION) CANDIDATE COMPOUNDS (3)

The research results (1) and (2) presented above indicate that the cancer cell growth inhibitory effects of retinol and RA are not mediated by RA receptors. Based on these results, we developed new anti-cancer drug candidate compounds.

RA is a powerful cell differentiation inducer that suppresses the growth of various cancer cells (anticancer agent). However, side effects such as RA resistance and impaired dark adaptation have been reported. It is noteworthy that N-(4-hydroxyphenyl) retinamide (4-HPR, Fenretinide), which is an aminophenol derivative of RA (Fig. 6), does not have a differentiation-inducing effect, yet it exhibits a stronger cancer cell growth inhibitory effect than RA.^10,15,45) Clinical trials were conducted for various cancers (bladder cancer, lung cancer, prostate cancer, lymphoma, neuroblastoma),^45–54) but these were interrupted due to side effects of dark adaptation disorder (Fig. 6). Therefore, clinical development of 4-HPR was discontinued in spite of the fact that it has strong anticancer effects. Since 4-HPR shows side effects that include dark adaptation disorder, dry skin, rash, etc.,^10,12) we undertook the development of compounds that retain the activity of 4-HPR but have reduced side effects.

Fig. 6. Structures of RA, 4-HPR, Aminophenols, and p-Alkylaminophenols with Various Chain Lengths

4.1. p-Alkylaminophenol Created by Structure–Activity Relationship Studies

4.1.1. Key Structure of 4-HPR’s Cancer Cell Growth Inhibitory Effect: Methyl Aminophenol

The structure of 4-HPR can be divided into four compounds; RA, aminophenol: 4-AP, acetaminophenol: p-AAP, and methylaminophenol: p-MAP (Fig. 6A). We hypothesized that the cause of the side effects may be due to the cyclohexene ring. Therefore, we investigated the growth inhibitory effects of the component structures on cancer cells. In HL60 cells, 1 µM p-MAP suppressed approximately 33%, while RA and 4-HPR at 1 µM suppressed approximately 29% and 28%, respectively.¹²⁾ At a concentration of 10 µM, p-MAP was the strongest among RA and three aminophenols, having cell proliferation inhibitory effects to the same extent as 4-HPR (approximately 99.7% for p-MAP, 99.6% for 4-HPR) (approximately 39% for RA, 21.8% for 4-AP, and 4% for p-AAP). Furthermore, while 4-HPR suppressed the growth of drug-resistant cancer cells, p-MAP also showed inhibitory activity for growth on RA-resistant HL60 (HL60R) cells and RA-resistant breast cancer (MCF-7/Adr^R) cells, similar to 4-HPR.¹²⁾ In addition, although p-MAP was slightly weaker than 4-HPR, p-MAP among four compounds was the most potent inhibitor of the proliferation of breast cancer (MCF-7) cells, liver cancer (HepG2) cells, and prostate cancer (DU-145) cells. From these results, it was clear that p-MAP has the same level of anticancer activity as 4-HPR, that p-MAP has the strongest cell proliferation inhibitory action among the four kinds of compounds, and that the methyl group is a key structure of this action.¹²⁾

In order to elucidate the action mechanism of p-MAP, DNA fragmentation-agarose analysis was performed using HL60 cells, and a fragment ladder was observed in the p-MAP and 4-HPR at a concentration of 10 µM. From the above results, we concluded that p-MAP and 4-HPR induced apoptosis and suppressed cell proliferation.¹²⁾

4.1.2. p-Alkylaminophenol with Extended Methyl Chain

4.1.2.1. p-Butylaminophenol, p-Hexylaminophenol, and p-Octylaminophenol

Therefore, p-alkylaminophenol derivatives with extended polymethyl chains (p-butylaminophenol (p-BAP), p-hexylaminophenol (p-HAP), and p-octylaminophenol (p-OAP)) were synthesized (Fig. 6B), and their growth inhibitory effects on HL60 cells were investigated. As a result, we learned that the longer the chain length, the stronger the effect.^55,56)

4-HPR suppressed the proliferation of HL60 cells and RA-resistant HL60R cells in a concentration-dependent manner. In HL60 cells, p-OAP showed stronger cell proliferation inhibitory effects than 4-HPR at a low concentration of 0.1 to 1 µM. At a concentration of 4 µM, p-OAP showing approximately 60% inhibition was as active as 4-HPR, which showed approximately 62% inhibition. In contrast, in HL60R cells, 4 µM p-OAP showing approximately 70% inhibition was weaker than 4-HPR, which showed approximately 90% inhibition. p-OAP was effective against both HL60 and HL60R cells.⁵⁵⁾ In addition, p-OAP showed stronger proliferation inhibitory activity against RA-resistant breast cancer (MCF-7/Adr^R) cells than 4-HPR, but was equally effect against liver cancer (HepG2) cells or slightly less effective against breast cancer (MCF-7) cells. The cell proliferation inhibitory effects were enhanced depending on the alkyl chain length. In prostate cancer (DU-145) cells, p-BAP, p-HAP, and p-OAP were more potent than 4-HPR.⁵⁵⁾ These results indicate that p-OAP is the most effective among the five p-alkylaminophenols.

Whether these compounds induce apoptosis in HL60 cells was investigated by DNA fragmentation-agarose analysis. No fragmentation was observed in the DNA extracted from the control cells. However, a fragment ladder was observed at a concentration of 10 µM of p-alkylaminophenol. The ladder strength increased depending on the alkyl chain length. These results suggested that p-alkylaminophenol induces apoptosis and suppresses cell proliferation.

4.1.2.2. p-Dodecylaminophenol, p-Decylaminophenol, N-(4-Hydroxyphenyl) dodecanamide, and N-(4-Hydroxyphenyl) decanamide

More extended side chains of p-alkylaminophenol (p-decylaminophenol (p-DAP), p-dodecylaminophenol (p-DDAP)) (Fig. 6B) and p-acylaminophenol (N-(4-hydroxyphenyl) decanamide (4-HPD), N-(4-hydroxyphenyl) dodecanamide (4-HPDD)) (Fig. 6C) were synthesized and their effects on cancer cells were investigated (1.–5.).^57,58) It was found that p-DDAP showed growth inhibitory effects greater than 4-HPR (Figs. 6A, B). On the other hand, in all cancer cells examined, acylaminophenol 4-HPDD and 4-HPD showed weaker effects than the alkylaminophenols, p-DDAP and p-DAP (Figs. 6B, C).

1. p-DDAP and p-DAP suppressed the proliferation of HL60 and HL60R cells at lower concentrations than 4-HPR.⁵⁷⁾

2. The activity of p-DDAP was not limited to leukemia cells. In MCF-7 cells, p-DDAP and p-DAP showed growth inhibitory effect to the same extent as 4-HPR. Furthermore, for RA-resistant MCF-7/Adr^R cells, p-DDAP and p-DAP suppressed cell proliferation by almost 100%, while 4-HPR exhibited approximately 60%.⁵⁸⁾

3. In drug-resistant prostate cancer cells PC-3, the cell proliferation inhibitory effect became stronger with the elongation of the polymehtyl chain of alkylaminophenol (Fig. 7A (a)). In addition, acylaminophenol (Fig. 7A (b)) showed only a weak effect. Therefore, p-DDAP with an alkyl chain length of 12 showed the strongest effect. p-DDAP arrested cell cycle in S phase and induced apoptosis by reduction of bcl-2 mRNA and activation of caspase-3.⁵⁹⁾

Fig. 7. (A) Growth Inhibitory Effects of p-Alkylaminophenol on Prostate Cancer Cells and (B) Effects of p-DDAP on Blood Retinol Concentration

Next, the in vivo effect of p-DDAP was examined using nude mice. When 4-HPR and p-DDAP (15 mg/kg) were administered to PC-3 cell-bearing mice, both compounds significantly reduced the tumor volume. When the administration methods were intraperitoneal (i.p.) and intravenous (i.v.), the tumor volume reduction effects of p-DDAP were modest in both cases.⁵⁹⁾

4. When the growth inhibitory and apoptosis-inducing effects of p-DDAP on neuroblastoma (NB-39-nu) cells, which are associated with pediatric malignant tumors, were examined, the growth inhibitory effects on NB-39-nu cells exhibited potencies in the order of p-DDAP, 4-HPR, and RA at a concentration of 4 µM. p-DDAP arrested the cell cycle of NB-39-nu cells in the G₀/G₁ phase, induced DNA fragmentation, reduced the apoptosis marker bcl-2 mRNA, and induced caspase-3 and caspase-8 activation.⁶⁰⁾

The growth inhibitory effects of p-DDAP on other neuroblastoma (SK-N-AS cells (non-N-myc amplification), IMR-32 cells (N-myc amplification)) were also potent in the order of p-DDAP, 4-HPR, and RA.¹⁴⁾ In SK-N-AS cells, p-DDAP induced apoptosis mediated by decreased expression of bcl-2 mRNA and activation of caspase-3, and suppressed proliferation. In contrast, in IMR-32 cells p-DDAP showed induction of apoptosis through a decrease in the expression of bcl-2 mRNA and growth inhibitory effects by the decreases in the expression of N-myc mRNA. In vivo, the tumor volume of SK-N-AS cancer-bearing mice was significantly reduced.¹⁴⁾

5. When the effects of p-DDAP, RA, and 4-HPR on pancreatic cancer (MIA Paca2) cells and cholangiocarcinoma (HuCCT1) cells, which are refractory cancers with low 5-year survival rate were examined, RA had little effect on the proliferation of both cells. 4-HPR strongly suppressed the proliferation of MIA Paca2 and HuCCT1 cells, and their IC₅₀ values were 7 and 6 µM, respectively. In addition, p-DDAP and p-DAP suppressed the proliferation of both cells at lower concentrations to greater extents than 4-HPR, with the IC₅₀ values of p-DDAP for MIA Paca2 and HuCCT1 being 10 and 2 µM, respectively.⁶¹⁾ Since both cells had an activating mutation of the KRAS gene (indicator of malignancy), it was found that p-DDAP is also effective against highly malignant refractory cancer. Pancreatic cancer (MIA Paca2) cells and cholangiocarcinoma (HuCCT1) cells, which are refractory cancers, showed a growth inhibitory effects through suppressing the MEK/ERK pathway and PI3K/Akt pathway rather than inducing apoptosis.⁶¹⁾

4.2. Side Effects of p-DDAP and Effective Cancer Cells

4.2.1. Effect of p-DDAP on Blood Retinol Concentration

Next, the effects of p-DDAP on blood retinol concentrations, which are closely related to side effects, was investigated. As shown in Fig. 7B, 4-HPR significantly reduced blood retinol levels, whereas p-DDAP did not affect blood retinol levels.⁵⁹⁾ Therefore, it was concluded that p-DDAP can eliminate RA resistance and dark adaptation disorder, which are side effects of 4-HPR, and could be more powerful anticancer drugs than either RA or 4-HPR without side effects.

4.2.2. Human Cancer Cells in Which p-DDAP Exerts a Cell Proliferation Inhibitory Effect

p-DDAP had cell proliferation inhibitory effects on 14 types of human cancer cells including refractory and drug-resistant and sensitive cancer types.¹⁵⁾ In addition, as mentioned earlier, it was confirmed that p-DDAP decreases tumor volume of prostate cancer PC-3 cells and neuroblastoma SK-N-AS cancer cells when examined in vivo. Furthermore, in order to investigate the effects of p-DDAP on invasion and metastasis of cancer cells, a matrigel infiltration test was conducted using highly invasive fibrosarcoma (HT1080) cells. p-DDAP and 4-HPR significantly reduced the number of infiltrating cells by approximately 80 and 20%, respectively, while RA did not change the number of infiltrating cells.⁶²⁾ In addition, p-DDAP significantly reduced the secretion of matrix metalloproteinase-9 (MMP-9), significantly suppressed the expression of MMP-9 mRNA, and increased expression of inducing cysteine-rich protein with Kazal motif’s (RECK) proteins and mRNAs, which negatively regulate the expression of MMP-9 mRNA. Therefore, we concluded that p-DDAP suppresses the invasion and metastasis of cancer cells by suppressing the expression of MMP-9.

4.2.3. Comparison of the Anticancer Effects of p-DDAP and Retinol or RA

Table 2 shows a comparison of p-DDAP and retinol or RA on cancer cell growth inhibitory effects, binding to RA receptors, and mechanism of action. While retinol is effective against insensitive cancers, including refractory cancers and drug-resistant cancers, and RA is effective against sensitive cancers, p-DDAP was found to have both properties. p-DDAP is effective against both sensitive and insensitive cancers. In addition, p-DDAP and retinol show low binding to the RA receptor,⁶³⁾ whereas RA does. Furthermore, p-DDAP is also characterized in its mechanism of action, unlike retinol and RA. Since p-DDAP is extremely effective against many cancers while having few side effects, clinical applications of p-DDAP are anticipated.

Table 2. Comparison of Actions of p-DDAP, Retinol and RA

	Growth inhibition		Induction of diffrentiation	Binding to RAR	Mechanisms
	Sensitive cancer cells	Insensitive cancer cells*
p-DDAP	+	+	−	−	< HL60 cell, etc. > Apoptosis < refractory cancer > MEK/ERK pathway, PI3K/Akt pathway
Retinol	−	+	−	−	< refractory cancer > ER stress, Autophagy
RA	+	−	+	+	< HL60 cell, etc. > RAR, RXR, Retinoylation

*Drug resistant or refractory cancer cells.

Modified with permission from table of Pharmacol. Ther., 230, 107942 (2022).

4.3. Summary—p-DDAP

As described above, while using 4-HPR as a lead compound, we were able to develop p-DDAP as an anti-cell proliferative with greater efficacy than 4-HPR.

Specifically,

1. 1.    p-DDAP had in vitro growth inhibitory effect on PC-3 cells, which were drug-resistant cancers, and was the most potent among 4-HPR and p-alkylaminophenols with side chains of various lengths.
2. 2.    p-DDAP reduced the tumor volume of PC-3 cell-bearing mice in vivo, and also reduced the tumor volume by changing the administration method, i.p. or i.v. administration.
3. 3.    p-DDAP eliminates the dark adaptation disorder, which is a side effect of 4-HPR, since p-DDAP does not affect the blood retinol concentration.
4. 4.    p-DDAP is effective not only for sensitive cancer but also for insensitive cancer such as refractory cancer.
5. 5.    p-DDAP does not show toxicity to normal cells or mice. Since p-DDAP has phenolic OH, it may be metabolized by glucuronidation and sulfate conjugate.

From these facts, p-DDAP represents an effective compound against refractory cancer, which was difficult to develop in the past; it is not toxic to normal cells and mice; it is easily metabolized by conjugation reaction and it has few side effects. Therefore, we have concluded that p-DDAP is a promising anticancer drug candidate in the early stages of advancing clinical trials.

5. CONCLUSION

As mentioned above, our achievements are as follows:

1. 1.    Discovery of anti-refractory cancer action of retinol and elucidation of its mechanism of action
2. 2.    Development of a new method to measure retinoylated proteins
3. 3.    Elucidation of cell mechanisms of proliferation inhibitory actions by retinoylated proteins
4. 4.    Development of new anticancer drug candidates

Vitamin A is a miracle nutrient that exhibits a wide range of physiological activities throughout the body. In these studies, we demonstrated that all types of vitamin A play an important role in the body. In particular, RA, which is an active constituent of vitamin A, is a compound involved in cell proliferation and differentiation, and therefore it has been involved in the onset and treatment of cancer. However, it was recently found that retinol, which is the most abundant in the circulatory system among vitamin A, has anticancer effects on refractory cancers for which RA has no effect. This strongly suggests that ingesting foods containing vitamin A to avoid vitamin A deficiency and maintain vitamin A concentration may lead to prevention and treatment of cancer. In developing countries, many infants deficient in vitamin A have lost their lives due to the inability to supplement vitamin A. Promoting nutritional management of vitamin A, maintaining health, recovering from illness and saving lives is a global challenge.

In retinoylation, post-translational modification by RA, retinoylated proteins (enzymes, structural proteins, etc.) have been sequentially identified using a novel detection method. Nuclear proteins, including various factors responsible for controlling the expression of genes and proteins phosphorylated by retinoylated PKA in RA-dependent manner, have been identified and found as new molecular targets. While both the retinoylated protein and RA receptors have high affinity for RA, it is important to note that the fashions in which properties of the proteins (stability, localization, activity, etc.) are affected by retinoylation are different from RA receptor. Examination of ligand binding sites and structural and functional changes of identified modified proteins is needed to elucidate new epigenetic signaling pathways consisting of histone modification reactions, such as retinoylation and phosphorylation.

By conducting research on vitamin A, we would like to find new targets, develop improved new anticancer agents such as p-DDAP, connect them to clinical trials, and aim to establish treatment/prevention methods. Since vitamin A is a panacea for many diseases other than cancer, the development of new drugs based on the mechanisms of action of vitamin A can be anticipated.

Acknowledgments

I am extremely honored and grateful for having received the “The Pharmaceutical Society of Japan Award for Divisional Scientific Contribution.” In particular, I would like to express my deepest gratitude to Dr. Atsushi Ichikawa, Professor Emeritus of Kyoto University, Drs. Theodore R. Breitman and Terrence Burke, Jr. of National Institutes of Health, National Cancer Institute, U.S.A., Dr. Tetsuya Fukui, Professor Emeritus of Hoshi University, and Professor Kazuhisa Nakayama of the Faculty of Pharmaceutical Sciences, Kyoto University for their support and guidance. I would like to express my sincere gratitude to Chairman Takuo Otani, directors, successive presidents, and vice presidents of Hoshi University for providing a place for research and education and the support to conduct this research. I appreciate the cooperation of Dr. Masahiko Imai, Daisuke Saito, M.S., Dr. Masahiro Yamasaki, Dr. Shinya Hasegawa, Dr. Katsuhiko Takahashi, Dr. Chuan Li, Dr. Yoshinori Kubo, Dr. Akiyo Iwahori, Toshihiro Ohba, M.S., Yusuke Watanabe, M.S., Asako Sakai, M.S. of Hoshi University, Dr. Koji Tamura of Tohoku University, Dr. Shioko Kimura, Dr. Charis Liapi, Dr. Wayne B. Anderson, Janet M. Hauser, M.S. of National Institutes of Health, U.S.A., Dr. Karry R. Norum, Dr. Anne M. Myhre, and Dr. Rune Blomhoff of Oslo University, Norway, Dr. William S. Blaner of Columbia University, U.S.A., and Richard C. Moon of Chicago University, U.S.A. Finally, I would like to express my deep gratitude for having been able to conduct this research under the support of the Ministry of Education Science Research Fund, the Hoshi University Otani Memorial Research Grant, and the Sankyo Foundation of Life Science.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2022 Pharmaceutical Society of Japan Award for Divisional Scientific Contribution.

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