Mono-Carbonyl Curcumin Analogs for Cancer Therapy

Abstract

Curcumin has long been recognized for its anti-inflammatory properties. An antitumor effect has been recently reported in curcumin and clinical trials are being conducted. However, a large amount of required intake to obtain the antitumor effect of curcumin has been regarded as a problem. Therefore, curcumin analogs have been created by many researchers to enhance the effects of curcumin. We have synthesized >50 curcumin analogs and revealed greater growth suppression of various tumor cells with mono-carbonyl analogs than curcumin. Mechanistically, mono-carbonyl analogs inhibited transcriptional activity (e.g., nuclear factor kappa B, signal transducer, and activator of transcription 3) or activated caspase-3. Additionally, mono-carbonyl analogs of curcumin control tumor cell metabolism. Herein, we summarize the current knowledge about mono-carbonyl curcumin analogs and discuss their potential clinical applications.

1. INTRODUCTION

Curry spice “turmeric” used for school lunches and dining tables was discovered as a medicinal plant in India approximately 4000 years ago. Turmeric, which is yellow and has various medicinal properties, has been introduced in the travels of Marco Polo in the 13th century. Curcumin, which is a substance in turmeric (Curcuma longa), has multiple potentials, including antioxidant, anti-inflammation, and anticancer effects.¹⁾ Turmeric arrived in China around 700 AD and in East Africa around 800 AD. Additionally, turmeric is introduced as a plant similar to saffron in the Travel of Marco Polo, a book written by Marco Polo, who is an Italian adventurer in the 13th century.²⁾ Turmeric has different names in India, including Haldi in the North and Manar in the South. The etymology of turmeric comes from the Latin terramelita (contribution to the earth), referring to the yellow-resembling minerals containing minerals. It has >50 Sanskrit names, including names referring to effects, such as Jawarantika (fever cure) and jayanti (fighting disease), and color names such as kanchani (golden) and pinga (reddish brown).²⁾

Turmeric is the product of Curcuma longa, which is a rhizomatous perennial plant belonging to the Zingiberaceae family. Curcuma has >130 species globally.³⁾ Curcuma is a fun ornamental plant, with red, white, and pale orange flowers. Turmeric is reported to contain >100 ingredients. Its main components in a standard form are volatile oils (<3.5%), including turmerone, which is responsible for the aroma, and curcumin (approximately 5–6.6%), which is responsible for the color.⁴⁾ Additionally, curcumin is recognized as a natural antioxidant and was first purified and extracted in 1842. To date, more than 5000 research reports have been published on curcumin, and various effects, such as anti-inflammatory and anticancer effects, have been clarified.

A clinical trial of curcumin intake has revealed no toxic effects on humans up to a dose of 8000 mg/d for 3 months.⁵⁾ Curcumin intake at 4000 mg/d for 30 d prevents aberrant crypt foci in colorectal neoplasia (Clinical trial Phase II).⁶⁾ Turmeric contains approximately 2–8% curcumin; therefore, ≥2 kg of turmeric should be consumed to achieve its anticancer effects. The average intake of turmeric is approximately 2000–2500 mg in India.⁷⁾ The low bioavailability of curcumin is due to its chemical and biological instability under physiological conditions.⁸⁾ To overcome this problem, people focused on the development of formulation methods, prodrugs, and synthetic derivatives of curcumin (e.g., di-carbonyl curcumin analogs including dimethoxy-curcumin). In particular, mono-carbonyl analogs, in which the two carbonyl groups of curcumin are reduced to one, have attracted much attention, since the large contribution of the highly reactive 1,3-dicarbonyl structure to the degradation of curcumin.⁹⁾ This review summarizes current knowledge on curcumin-derived mono-carbonyl curcumin analogs, in particular their antitumor effects.

2. SYNTHESIS OF MONO-CARBONYL CURCUMIN ANALOGS

Curcumin has β-diketone moiety, which contributes to poor bioactivity.¹⁰⁾ Therefore, mono-carbonyl analogs of curcumin have been synthesized to improve bioactivity (Chart 1).

Chart 1. Structure of Mono-Carbonyl Curcumin Analogs

σ-Symmetric 1,5-diaryl-3-oxo-1,4-entadiens, GO-Y022 and GO-Y030, were synthesized by double aldol condensation reactions of the corresponding aromatic aldehydes 2 or 4 with acetone under the basic conditions (Fig. 1: Chart A). The phenolic hydroxyl group of vanillin (1) was temporarily protected by an ethoxyethyl group, which was removed by hydrochloric acid treatment after the aldol reaction, in GO-Y022 synthesis. GO-Y030 precursor 4 was prepared from 3,5-dihydroxybenzaldehyde by the methoxymethylation in 65% yield.¹¹⁾

Fig. 1. Synthesis of Mono-Carbonyl Curcumin Analogs

Synthesis of asymmetric 1,5-diaryl-3-oxo-1,4-entadien, GO-Y078, started with aldol condensation of 3,4,5-trimethoxybenzaldehyde (5) and acetone with the help of dimethylammonium dimethylcarbamate in 89% yield (Fig. 1: Chart B). The resulting methyl ketone 6 was subjected to a second aldol condensation with ethoxyethyl-protected arylaldehyde 7 followed by deprotection under acidic conditions to yield GO-Y078.¹²⁾

The Snyder and Shoji group developed 3,5-bis(arylmethylidene)-4-peperidones, EF-24, EF-31, and USB109 (Fig. 1: Chart C). EF-24 was obtained as its AcOH salt from 4-piperidone hydrochloride monohydrate (10) and aldehyde 9 by an aldol condensation reaction under acidic conditions. The aldol condensation of aldehyde 11 and the corresponding carbonyl derivatives (hydrate 10 for EF31, ketone 12 for USB109) (Fig. 1: Chart D) under the basic conditions afforded EF-31 and USB109 in 69 and 97% yields, respectively, using cetyltrimethylammonium chloride (CTACl) as the phase transfer catalyst.¹³⁾

3. GO-Y022

GO-Y022 (also known as FLLL11 or Deketomin^®), 1,5-bis(4-hydroxy-3-methoxyphenyl)-1,4-pentadiene-3-one, is a diarylpentanoid analog (C5) and is generally generated when cooking curry.¹⁴⁾ GO-Y022 demonstrated an antitumor effect in the case of MDA-MB-451 breast tumor cells but does not inhibit the human epidermal growth factor receptor and AKT pathway, which contribute to drug resistance, invasion, and angiogenesis.¹⁵⁾ Yoshida et al. attempted feeding a high fat diet 32 with 0.5% (w/w) GO-Y022 in mice and found that no toxic effects (no increasing serum level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, creatinine and lactate dehydrogenase (LDH) compared with high fat diet 32 alone) and below detection limits in the kidney, liver, blood, and spleen.¹⁶⁾ Additionally, a high fat diet 32 with 0.5% (w/w) GO-Y022-feeding for 7 weeks prevents oncogenesis in Gan mice (K19-Wnt1/C2 mE mice). Mechanistically, 5 µM of GO-Y022-treatment prevents signal transducer and activator of transcription 3 (STAT3) activation in tumor cells (Human gastric tumor cell lines; SH-10-TC and GCIY) in vitro,¹⁶⁾ which can contribute to reduced tumor cell proliferation, survival, and metastasis.¹⁷⁾ Another report revealed that GO-Y022 treatment (<3.1 µM) increased caspase-3 activity and Bcl-2 expression in lung tumor cells (NCI-H520 and NCI-H23) in a dose-dependent manner.¹⁸⁾ Conversely, we demonstrated that 5 µM of GO-Y022-treatment can promote GCIY and SH-10-TC cell metabolism (e.g., more glycolysis and L-lactate production) in vitro and Foxp3⁺Tregs’ generation from naïve CD4⁺ T cells in the co-cultured with SH-10-TC. Therefore, the combination of GO-Y022 and 2-deoxy-glucose (a glycolysis inhibitor) can effectively induce gastric tumor cell death and prevent Foxp3⁺Tregs’ generation in the tumor microenvironment¹⁹⁾ (Fig. 2). Another report showed that protein–protein interaction network of all differentially expressed proteins in U-87 MG cells (malignant glioma) have revealed that 10 µM of GO-Y022 treatment upregulated metabolic enzymes ENO1 and TPI1 meaning helps glycolysis.²⁰⁾ Thus, GO-Y022 has an ability to promote glycolysis of gastric tumor cells and malignant glioma cells, which will affect anti-tumor immunity in the tumor microenvironment via production of tumor metabolites.

Fig. 2. GO-Y022 Controls Metabolisms of Gastric Tumor Cells

Curcumin analog GO-Y022 treatment enhanced glycolysis and TGF-β/L-lactate production from gastric tumor cells, playing important roles in Tregs’ generation. A glucose analog, 2-deoxy-glucose, is taken up by gastric tumor cells upon GO-Y022 treatment and may inhibit cell survival and TGF-β/L-lactate production. Therefore, the co-administration of GO-Y022 and 2-deoxy-glucose is a powerful new strategy for antitumor immunity.

4. GO-Y030

GO-Y030 (also known as MS13) has a 5-carbon space between 3,5-bis(methoxymethoxy) phenol. GO-Y030 demonstrates higher cancer cell growth suppressive ability than curcumin and GO-Y022 because GO-Y030 strongly reduces the S-phase fraction (DNA synthesis) and induces apoptosis through the caspase-3 activation (e.g., 2.5 µM GO-Y030-treatment induced cleaved caspase-3 in colorectal tumor HCT116 and SW480 cells in vitro, but not in 2.5 µM curcumin-treatment or 2.5 µM GO-Y022-treatment)).^11,21) Not only colorectal carcinomas but also breast and pancreatic carcinoma cell growth can be inhibited by GO-Y030, strongly²²⁾ (Table 1).

Table 1. Growth Inhibitor of Curcumin Analogs in Human Tumor Cell Lines

Tumor cell	50% Growth inhibitor	Reference
KMS12-BM (Myeloma)	Curcumin: 10.3 µM	Kudo et al. 201136,37)
	GO-Y030: 1.50 µM
	GO-Y078: 0.60 µM
U266 (Myeloma)	Curcumin: 17.8 µM
	GO-Y030: 2.10 µM
	GO-Y078: 1.20 µM
MDA-MB-453 (Breast cancer)	Curcumin: >50.0 µM	Lin L et al. 200915)
	GO-Y022: 4.7 µM
	GO-Y078: 1.3 µM
MCA-MB-231 (Breast cancer)	Curcumin: 25.6 µM
	GO-Y022: 2.80 µM
	GO-Y078: 2.70 µM
GCIY (Gastric tumor)	Curcumin: 30.4 µM	Yoshida et al. 201816)
	GO-Y022: 7.32 µM
KATO-III (Gastric tumor)	Curcumin: 24.8 µM
	GO-Y022: 5.98 µM
SW480 (Colorectal cancer)	Curcumin: 10.2 µM	Cen et al. 200921)
	GO-Y022: 1.64 µM
	GO-Y030: 0.51 µM
	GO-Y078: 1.17 µM
A549 (Epitherial carcinoma)	Curcumin: 9.20 µM	Haque et al. 201559)
	GO-Y078: 1.61 µM
H1299 (Lung carcinoma)	Curcumin: 6.07 µM
	GO-Y078: 0.78 µM
MDA-MB-231 (Breast cancer)	Curcumin: 11.1 µM	Adams et al. 200413)
	EF24: 0.80 µM
RPMI7951 (Melanoma)	Curcumin: 11.1 µM
	EF24: 0.70 µM
TU212 (Laryngeal carcinoma)	EF24: 8.00 µM	Zhu et al. 201244)
	EF31: 7.00 µM
MIA PaCa-2 (Pancreatic tumor)	Curcumin: ≈ 10.0 µM	Nagaraju et al. 201354)
	EF31: ≈ 0.75 µM
	UBS109: ≈ 0.25 µM
HCT116 (Colorectal cancer)	Curcumin: 25.0 µM	Rajitha et al. 201658)
	EF31: 7.50 µM
	UBS109: 0.62 µM
HT-29 (Colorectal cancer)	Curcumin: 20.0 µM
	EF31: 2.50 µM
	UBS109: 2.50 µM

A concentration of 50% of growth inhibitory effects revealed a stronger antitumor effect of mono-carbonyl curcumin analogs than curcumin.

Many types of carcinogenesis show constitutive active nuclear factor kappa B (NF-κB) and STAT3, which is considered to contribute to carcinogenesis survival.^23,24) One of the mechanisms is that NF-κB and STAT3 induce the antiapoptotic Bcl2 family members through escape from apoptosis in cancer cells.^25–27) GO-Y030 can inhibit NF-κB and STAT3 activation in cancer cells^22,28,29) and therefore also strongly induced apoptosis and inhibit the growth of many types of carcinomas. Another report has revealed that GO-Y030 markedly reduced proto-oncogene c-Myc in HTC116 (a human colon cancer cell line) and prevented their cell growth.³⁰⁾ A model of intestinal cancer revealed a significantly prolonged lifespan in APC(580D/+) mice that are fed a 0.5% GO-Y030 diet than those fed the basal diet.³¹⁾ We observed that 0.5% GO-Y030 diet-fed APC(580D/+) mice demonstrated skewing intestinal cancer without any biological side-effect. Currently, we find that 0.25 µM of GO-Y030-treatment inhibits the generation of Foxp3⁺Tregs in response to transforming growth factor beta (TGF-β) through the inhibition of p300-induced NF-κB activity in vitro.³²⁾ Additionally, 0.25 µM GO-Y030-treatment inhibits the stability of Foxp3⁺Tregs (less Foxp3 expression and de-methylation status of conserved non-cording sequence 2 of Foxp3 in cultured CD4⁺CD25⁺Tregs for 18 h in vitro). In vivo study also demonstrated that 5 mg/kg GO-Y030-treatment (intraperitoneal (i.p.) injection, each day from day 7 after 2.5 × 10⁵ B16-F10 melanoma cells subcutaneously injection) reduced the size of tumor and the percentages of Foxp3⁺Tregs in CD4⁺ T cells in tumor microenvironment.³²⁾ GO-Y030 has multi-potent cancer therapy because Foxp3⁺Tregs prevent antitumor immunology.³³⁾ Additionally, the 5 mg/kg of GO-Y030-treatment boosts cancer immunotherapy using an anti-programmed cell death-1 (PD-1) antibody (i.p. injection, 200 µg/mice every 3 d from day 7 after 2.5 × 10⁵ B16-F10 melanoma cells subcutaneously injection).³⁴⁾ Additionally, we revealed that 3.125 µM GO-Y030-treated B16-F10 melanoma cells (24 h, in vitro) were significantly reduced glycolysis compared with dimethyl sulfoxide (DMSO)-treated B16-F10 melanoma cells (Control)³⁴⁾ (Fig. 3), which plays important roles in tumor cell proliferation and survival.³⁵⁾ Thus, the curcumin analog GO-Y030 can inhibit glycolysis of melanoma cells and Tregs’ generation in melanoma microenvironment.

Fig. 3. GO-Y030 Inhibits Melanoma Cell Metabolism

Curcumin analog GO-Y030 can prevent Tregs’ generation in the tumor microenvironment. GO-Y030 treatment also inhibits glycolysis and TGF-β production from melanoma cells. One of the mechanisms revealed that glucose transporter (Glut 1), hexokinase 1 (Hk1: an enzyme catalyzes the first step in glycolysis), and oxidative ATP production in melanoma cells have reduced in the presence of GO-Y030, which contributed to tumor cell death.

5. GO-Y078

GO-Y078 is 1,5-diaryl-3-oxo-1,4-pentadienes analogs, similar to GO-Y030. The solubility (1.08 mg/L in Deuterium-depleted water) is approximately four times higher than GO-Y030 (0.26 mg/L in Deuterium-depleted water) and more than twice that of curcumin (0.54 mg/L in Deuterium-depleted water).³⁶⁾ Two micromolar GO-Y078-treated myeloma cells induces apoptosis of cancer cells and inhibits NF-κB and STAT3 activation more strongly than 2 µM curcumin-treatment (24h, in vitro).³⁷⁾ In addition, myeloma cells (KMS12-BM) was treated 2 µM GO-Y078 showed less MYC expression, which would be contribute to inhibit glycolysis.³⁷⁾ Thus, GO-Y078 has a more potent antitumor effect compared with curcumin. In contrast, GO-Y078 and GO-Y030 do not induce apoptosis of non-carcinoma cells at doses as high as 50 µM.³⁶⁾ In vivo situation, peritoneal carcinoma apoptosis of gastric cancer mice model (5 × 10⁶ human gastric tumor GCIY cells into the abdominal cavities 6-week-old male KSN Slc mice at day 0) shows that i.p. administration of GO-Y078 (133  or 266 mg/kg doses at day 7 and 14) significantly extended to survival span than that of control mice, but not GO-Y030 (155 mg/kg doses at days 7 and 14). Additionally, 1.2 µM of GO-Y078-treatment effectively induced apoptosis, growth inhibition and wound hearing inhibition of Human Umbilical Vein Endothelial Cells (HUVEC) in vitro.³⁸⁾ Using Xenopus laevis tadpole, 6.0 µM GO-Y078-treatment showed inhibition of vasculogenesis.³⁸⁾ Thus, GO-Y078-treatment inhibits tumor angiogenesis through actin stress fiber formation inhibition.³⁸⁾ GO-Y078 overcomes the resistance of angiogenesis by vascular endothelial growth factor (VEGF) inhibitors.³⁹⁾ The reason is that GO-Y078 dose-dependently downregulates fibronectin 1 (a key regulator for tumor angiogenesis) in VEGF-resistant HUVEC cell line.³⁹⁾ Thus, GO-Y078 induced apoptotic cell death via inhibition of NF-κB/STAT3 activation and tumor angiogenesis; however it is still unclear whether GO-Y078 controls metabolisms of tumor cells and anti-tumor immunity.

6. EF24

EF24 ((3E,5E)-3,5-bis[(2-fluorophenyl) methylene]-4-piperidinone) was first synthesized by Dr. Shoji’s group in 2004 and shows that the mean growth inhibitory concentration of EF24 was less than half of the curcumin in melanoma RPMI7951 and Breast MDA-MB-231.¹³⁾ The grown inhibitory concentration of EF24 is significantly less than that of curcumin in adrenal carcinoma cells^40,41) (Table 1). Interestingly, EF24 (i.p. injection daily, 200 µg/kg body weight) inhibits the size of tumor and angiogenesis in colon cancer HCT-116 xenograft using 5-week-old male athymic nude mice in vivo. Mechanistically, 1 µM/L of EF24-treatment decreased VEGF and interleukin (IL)-8 expression in HCT-116 cells in vitro, which are potent inducers of angiogenesis in tumor microenvironment.⁴²⁾ Thus, EF24 induced apoptosis in many types of cancer cells.^43,44) For example, liver cancer PLC/PRF/5 and Hep3B cells, 2 µmol/L EF24-treatment in vitro showed that these liver cancer cells significantly induced apoptotic cell death compared with DMSO-treated (control). Using xenograft model (2 × 10⁷ PLC/PRF/5 cells injected to the flank of nude mice at day 0), i.p. injection of 20 mg/kg EF24 in each day (from day 7) suppressed tumor growth. Additionally, a combination of EF24 with 7-ethyl-10-hydroxycamptothecin (SN38), which is a type 1 topoisomerase inhibitor, induced enhancement to colon cancer growth suppression.⁴⁵⁾ Briefly, 0.5 × 10 colon26 cancer cells subcutaneously inoculated in hind paw at day 0. Seven days later, 50 µL of SN38 (5 mg/mL) and EF24 (200 µg/mL) were injected subcutaneously under the tumor in each day. Another 22 d later, the size of tumor in SN38/EF24-treated mice were less than 10 times compared with non-treated mice (control). Mechanistically, EF24-treatment inhibits NF-κB signal pathway (a key regulator of tumor development) in PLC/PRF/5 cells both in vivo and in vitro.^43,44) In human prostate cancer DU145 cells, 5 µM EF24-treatment also blocks activation of NF-κB, but not the JAK–STAT signal pathway in vitro in human prostate cancer DU145 cells.^46,47) Using xenograft model (10⁶ DU145 cells injected into the franks of NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ mice at day 0), EF24-treatment (200 µg/kg, i.p. injection in each day from day 7) prevented the tumor growth and reduced NF-κB-target genes’ expression (e.g., CDK4 and CDK6) in the tumor side.^46,47) In human ovarian CR cancer cell, 2 µM EF24-treatment induced the phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) and enhanced tumor suppressor protein p53 expression within 3 h in vitro in human ovarian CR cancer cells.⁴⁸⁾ Other reports have revealed that reactive oxygen species (ROS) has been induced by 20 µM of EF24 treatment in DU-145 prostate cancer and MDA-MB-231 breast cancer or 7.5 µM EF24-treatment in SGC-7901 gastric cancer, and these cells showed inhibition of growth either in vitro cultures.^40,49,50) In vivo xenograft model (Subcutaneously injected 5 × 10⁶ SGC-7901 into a right flank of athymic mice) showed that reduced the size of tumor and increase lipid peroxidation product MDA (a maker of ROS) in the tumor tissue when the mice were treated 3 mg/kg EF24 (i.p. injection) once every other day.⁵⁰⁾ In case human leukemia K562 cells, catalase-treatment prevents 2 µM EF24-treatment induced ROS expression, however the percentage of EF24-induced apoptotic cell death were no difference in the presence or absence of catalase in vitro.⁵¹⁾ Thus, EF24-induced ROS activation and ROS-induced apoptosis would be dependent on the type of cancer cells.

microRNAs (miRNAs) are single-stranded non-cording RNAs, which controls tumor growth and metastasis through a regulation of post-transcriptional gene expression. EF24 inhibits miR-21 and controls the miR-21 target gene PTEN as a tumor suppressor gene in human prostate cancer cells.⁴⁷⁾ Furthermore, EF24-treated human prostate cancer cells downregulated oncogenic miRNAs (miR-21, miR-26a, miR-24, miR-29a, and miR-30b) as a proliferation enhancer. Another report has revealed that 1 µM of EF24 treatment induced miR-33b and their target gene mobility group AT-hook 2 (HMGA2) in human A375 melanoma cells in vitro, which play a key role for tumor cell migration.⁵²⁾ Therefore, EF24 suppresses the proliferation and migratory potential of tumor cells via multiple pathways including inhibition of NF-κB pathway or miRNAs expression.

7. EF31

EF31 (3,5-bis(2-pyridinylmethylidene)-4-piperidone) was also synthesized by Dr. Shoji’s group in 2004.¹³⁾ EF31 exhibited NF-κB inhibition and an antitumor effect against A2780 (human ovarian carcinoma cells) and MIA PaCa-2 (human pancreatic carcinoma cells) in vitro, which is a 10-fold increase than curcumin.⁵³⁾ The TU212 squamous cell carcinoma xenograft model demonstrated a significantly reduced tumor size with i.p. injection of 12.5 mg/kg of EF31 (daily for 5 d/week).⁴⁴⁾ Male ND4 Swiss Webster mice were given an i.p. injection of 12.5 mg/kg of EF31 for pharmacological kinetics and demonstrated that plasma concentrations were peak (1000 ng/mL) within 0.5 h and then rapidly degraded (terminal elimination half-life averaged 2.2 h). 0.75–1.25 µM EF31 treatment showed downregulate DNA methyltransferase-1 expression in pancreatic cancer PANC-1 in vitro (but not in 10–20 µM curcumin treatment), which was associated with silenced tumor suppressor proteins (p16 and E-cadherin).⁵⁴⁾ The activation of STAT3 is associated with prognosis in human colorectal cancer.⁵⁵⁾ Rajitha et al. demonstrated that EF31 inhibits VEGF and STAT3 activation in colorectal cancer HC116 (7.5 µM for 24 h in vitro) and HT-29 (2.5 µM for 24 h in vitro), which would be a rational approach for antiangiogenetic therapy in patients with colorectal cancer.⁵⁶⁾ Xenograft models either HC116 or HT-29 showed that EF31-treatment (twice weekly 25 mg/kg i.v. injection for 3 weeks) reduced VEGF expression and activation of STAT3 compared with control (DMSO-treatment). Thus, EF31 showed anti-tumor effects via inhibition of NF-κB and STAT3 signal pathways.

8. UBS109

USB109, (3E,5E)-1-methyl-3,5-bis(2-pyridinylmethylene)-4-piperidinone, has a very similar structure and antitumor effects with EF31.⁵⁴⁾ UBS109 downregulated DNMT-1 expression through NF-κB inhibition in pancreatic tumor cells, which tends to be a little stronger than EF31. Additionally, 0.25 µM UBS109-treated human pancreatic tumor cells (Mia-PaCa2 and PANC-1) showed significantly less growth factors’ expression (VEGF, cyclooxygenase (COX)-2 and TGF-β) than that of DMSO-treatment as a control in vitro. Xenograft models using Mia-PaCa2 also showed that i.v. doses weekly with 25 mg/kg UBS109 significantly reduced growth factors’ expression (VEGF, COX-2, and TGF-β) in the subcutaneous tumor than DMSO treatment.⁵⁷⁾ Additionally, the effect was stronger than EF31 treatment. NF-κB contributes to the survival pathway (ERK and AKT) and cell cycle molecular expression (Cyclin D and E2F1) in colorectal tumor cells and it can be inhibited by UBS109 and EF31 treatment.⁵⁸⁾ Xenograft model using colorectal tumor cells (HCT116 and HT-29) revealed that UBS109 (twice weekly 25 mg/kg i.v, injection for 3 weeks) in addition to antitumor drug 5-fluorouracil (once weekly 30 mg/kg i.v. injection for 3 weeks) + oxaliplatin (once weekly 5 mg/kg i.v. injection for 3 weeks) significantly inhibited tumor growth.⁵⁶⁾ The study demonstrated that UBS109 inhibits NF-κB downstream molecules’ expression (VEGF and COX-2) and activation of STAT3 in colorectal tumor HT-29 cells both in vitro (2.5 µM for 24h) and in vivo (HT-29 xenograft models: twice weekly 25 mg/kg i.v. injection for 3 weeks) experiments, which contributes to tumor growth and motility. Thus, UBS109 controls tumor growth and motility via NF-κB and STAT3 signal pathways in tumor cells.

9. CONCLUSION

Mono-carbonyl curcumin analog revealed antitumor effects, which are stronger than curcumin. As structural commonalities, mono-carbonyl curcumin analogs have carbonyl linker, which play key roles to show anti-tumor effects. NF-κB and STAT3 signal pathways contribute to tumor growth and motility, and it’s inhibited by using mono-carbonyl curcumin analogs, respectively. Since EF24 and EF31 have amines (nitrogen functional group), they are well soluble in water when salted. As like nanoparticle curcumin, improving of solubility contributes to enhance anti-tumor effects. Another mono-carbonyl curcumin analogs (UBS109, GO-Y022 and GO-Y078) also improved solubility compared with curcumin, but not GO-Y030. Additionally, mono-carbonyl curcumin analog has multiple potentials for antitumor therapy, including antitumor immunity. GO-Y030-treatment reduced Tregs’ population in skin tumor microenvironment, which would contribute to enhance anti-tumor immunity. However, GO-Y022-treatment did not reduce Tregs’ population in gastric tumor microenvironment. We also found that GO-Y078-treatment did not reduce Tregs’ population in skin tumor microenvironment (Unpublished data). In the metabolic view of tumor cells, GO-Y030 inhibited glycolysis of melanoma cells. Tumor metabolites TGF-β and L-lactate positively regulate immune suppression by Tregs in tumor microenvironment; therefore, inhibition of tumor metabolites by GO-Y030-treatment resulted in less Tregs’ population. On the other hands, GO-Y022 promoted glycolysis of gastric tumor cells and production of tumor metabolites, which is contribute to maintain Tregs population in gastric tumor microenvironment. Therefore, GO-Y030 has more potent the boost for immune checkpoint inhibitor including anti-PD-1. In the future, it is expected that clinical trials will be conducted on the antitumor effects of Mono-carbonyl curcumin analogues.

Conflict of Interest

Our study was partially supported by Akita Industrial/Academic/Governmental Research Project on Developing Innovations, Joint Research and Promotion Project on Creating New Technologies and Industries, and Yamasaki Spice Promotion Foundation.

REFERENCES

• 1) Hewlings SJ, Kalman DS. Curcumin: a review of its’ effects on human health. Foods, 6, 92 (2017).
• 2) Prasad S, Aggarwal BB. Turmeric, the Golden Spice: From Traditional Medicine to Modern Medicine. Herbal Medicine: Biomolecular and Clinical Aspects. (Benzie IFF, Wachtel-Galor S eds.) Boca Raton, FL (2011).
• 3) Mishra S, Verma SS, Rai V, Awasthee N, Arya JS, Maiti KK, Gupta SC. Curcuma raktakanda induces apoptosis and suppresses migration in cancer cells: role of reactive oxygen species. Biomolecules, 9, 159 (2019).
• 4) Ohshiro M, Kuroyanagi M, Ueno A. Structures of sesquiterpenes from Curcuma longa. Phytochemistry, 29, 2201–2205 (1990).
• 5) Cheng AL, Hsu CH, Lin JK, et al. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res., 21 (4B), 2895–2900 (2001).
• 6) Carroll RE, Benya RV, Turgeon DK, Vareed S, Neuman M, Rodriguez L, Kakarala M, Carpenter PM, McLaren C, Meyskens FL Jr, Brenner DE. Phase IIa clinical trial of curcumin for the prevention of colorectal neoplasia. Cancer Prev. Res. (Phila.), 4, 354–364 (2011).
• 7) Basnet P, Skalko-Basnet N. Curcumin: an anti-inflammatory molecule from a curry spice on the path to cancer treatment. Molecules, 16, 4567–4598 (2011).
• 8) Moballegh Nasery M, Abadi B, Poormoghadam D, Zarrabi A, Keyhanvar P, Khanbabaei H, Ashrafizadeh M, Mohammadinejad R, Tavakol S, Sethi G. Curcumin delivery mediated by bio-based nanoparticles: a review. Molecules, 25, 689 (2020).
• 9) Moreira J, Saraiva L, Pinto MM, Cidade H. Diarylpentanoids with antitumor activity: a critical review of structure–activity relationship studies. Eur. J. Med. Chem., 192, 112177 (2020).
• 10) Zhao C, Zhang Y, Zou P, Wang J, He W, Shi D, Li H, Liang G, Yang S. Synthesis and biological evaluation of a novel class of curcumin analogs as anti-inflammatory agents for prevention and treatment of sepsis in mouse model. Drug Des. Devel. Ther., 9, 1663–1678 (2015).
• 11) Ohori H, Yamakoshi H, Tomizawa M, Shibuya M, Kakudo Y, Takahashi A, Takahashi S, Kato S, Suzuki T, Ishioka C, Iwabuchi Y, Shibata H. Synthesis and biological analysis of new curcumin analogues bearing an enhanced potential for the medicinal treatment of cancer. Mol. Cancer Ther., 5, 2563–2571 (2006).
• 12) Yamakoshi H, Ohori H, Kudo C, Sato A, Kanoh N, Ishioka C, Shibata H, Iwabuchi Y. Structure–activity relationship of C5-curcuminoids and synthesis of their molecular probes thereof. Bioorg. Med. Chem., 18, 1083–1092 (2010).
• 13) Adams BK, Ferstl EM, Davis MC, Herold M, Kurtkaya S, Camalier RF, Hollingshead MG, Kaur G, Sausville EA, Rickles FR, Snyder JP, Liotta DC, Shoji M. Synthesis and biological evaluation of novel curcumin analogs as anti-cancer and anti-angiogenesis agents. Bioorg. Med. Chem., 12, 3871–3883 (2004).
• 14) Dahmke IN, Boettcher SP, Groh M, Mahlknecht U. Cooking enhances curcumin anti-cancerogenic activity through pyrolytic formation of “deketene curcumin”. Food Chem., 151, 514–519 (2014).
• 15) Lin L, Hutzen B, Ball S, Foust E, Sobo M, Deangelis S, Pandit B, Friedman L, Li C, Li PK, Fuchs J, Lin J. New curcumin analogues exhibit enhanced growth-suppressive activity and inhibit AKT and signal transducer and activator of transcription 3 phosphorylation in breast and prostate cancer cells. Cancer Sci., 100, 1719–1727 (2009).
• 16) Yoshida T, Maruyama T, Miura M, Inoue M, Fukuda K, Shimazu K, Taguchi D, Kanda H, Oshima M, Iwabuchi Y, Shibata H. Dietary intake of pyrolyzed deketene curcumin inhibits gastric carcinogenesis. J. Funct. Foods, 50, 192–200 (2018).
• 17) Zou S, Tong Q, Liu B, Huang W, Tian Y, Fu X. Targeting STAT3 in cancer immunotherapy. Mol. Cancer, 19, 145 (2020).
• 18) Wan Mohd Tajuddin WNB, Abas F, Othman I, Naidu R. Molecular mechanisms of antiproliferative and apoptosis activity by 1,5-bis(4-hydroxy-3-methoxyphenyl)1,4-pentadiene-3-one (MS13) on human non-small cell lung cancer cells. Int. J. Mol. Sci., 22, 7424 (2021).
• 19) MaruYama T, Miyazaki H, Lim YJ, Gu J, Ishikawa M, Yoshida T, Chen W, Owada Y, Shibata H. Pyrolyzed deketene curcumin controls regulatory T cell generation and gastric cancer metabolism cooperate with 2-deoxy-d-glucose. Front. Immunol., 14, 1049713 (2023).
• 20) Lee YQ, Rajadurai P, Abas F, Othman I, Naidu R. Proteomic analysis on anti-proliferative and apoptosis effects of curcumin analog, 1,5-bis(4-hydroxy-3-methyoxyphenyl)-1,4-pentadiene-3-one-treated human glioblastoma and neuroblastoma cells. Front. Mol. Biosci., 8, 645856 (2021).
• 21) Cen L, Hutzen B, Ball S, DeAngelis S, Chen CL, Fuchs JR, Li C, Li PK, Lin J. New structural analogues of curcumin exhibit potent growth suppressive activity in human colorectal carcinoma cells. BMC Cancer, 9, 99 (2009).
• 22) Hutzen B, Friedman L, Sobo M, Lin L, Cen L, De Angelis S, Yamakoshi H, Shibata H, Iwabuchi Y, Lin J. Curcumin analogue GO-Y030 inhibits STAT3 activity and cell growth in breast and pancreatic carcinomas. Int. J. Oncol., 35, 867–872 (2009).
• 23) Lee H, Herrmann A, Deng JH, Kujawski M, Niu G, Li Z, Forman S, Jove R, Pardoll DM, Yu H. Persistently activated Stat3 maintains constitutive NF-kappaB activity in tumors. Cancer Cell, 15, 283–293 (2009).
• 24) Fan Y, Mao R, Yang J. NF-kappaB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein Cell, 4, 176–185 (2013).
• 25) Karin M. Nuclear factor-kappaB in cancer development and progression. Nature, 441, 431–436 (2006).
• 26) Choi HJ, Lee JH, Park SY, Cho JH, Han JS. STAT3 is involved in phosphatidic acid-induced Bcl-2 expression in HeLa cells. Exp. Mol. Med., 41, 94–101 (2009).
• 27) Banerjee K, Resat H. Constitutive activation of STAT3 in breast cancer cells: a review. Int. J. Cancer, 138, 2570–2578 (2016).
• 28) Sato A, Kudo C, Yamakoshi H, Uehara Y, Ohori H, Ishioka C, Iwabuchi Y, Shibata H. Curcumin analog GO-Y030 is a novel inhibitor of IKKbeta that suppresses NF-kappaB signaling and induces apoptosis. Cancer Sci., 102, 1045–1051 (2011).
• 29) Pozdeyev N, Berlinberg A, Zhou Q, Wuensch K, Shibata H, Wood WM, Haugen BR. Targeting the NF-kappaB pathway as a combination therapy for advanced thyroid cancer. PLOS ONE, 10, e0134901 (2015).
• 30) Yamakoshi H, Kanoh N, Kudo C, Sato A, Ueda K, Muroi M, Kon S, Satake M, Ohori H, Ishioka C, Oshima Y, Osada H, Chiba N, Shibata H, Iwabuchi Y. KSRP/FUBP2 is a binding protein of GO-Y086, a cytotoxic curcumin analogue. ACS Med. Chem. Lett., 1, 273–276 (2010).
• 31) Shibata H, Yamakoshi H, Sato A, Ohori H, Kakudo Y, Kudo C, Takahashi Y, Watanabe M, Takano H, Ishioka C, Noda T, Iwabuchi Y. Newly synthesized curcumin analog has improved potential to prevent colorectal carcinogenesis in vivo. Cancer Sci., 100, 956–960 (2009).
• 32) MaruYama T, Kobayashi S, Nakatsukasa H, Moritoki Y, Taguchi D, Sunagawa Y, Morimoto T, Asao A, Jin W, Owada Y, Ishii N, Iwabuchi Y, Yoshimura A, Chen W, Shibata H. The curcumin analog GO-Y030 controls the generation and stability of regulatory t cells. Front. Immunol., 12, 687669 (2021).
• 33) Wing JB, Tanaka A, Sakaguchi S. Human FOXP3(+) regulatory T cell heterogeneity and function in autoimmunity and cancer. Immunity, 50, 302–316 (2019).
• 34) MaruYama T, Kobayashi S, Shibata H, Chen W, Owada Y. Curcumin analog GO-Y030 boosts the efficacy of Anti-PD-1 cancer immunotherapy. Cancer Sci., 112, 4844–4852 (2021).
• 35) Fischer GM, Vashisht Gopal YN, McQuade JL, Peng W, DeBerardinis RJ, Davies MA. Metabolic strategies of melanoma cells: mechanisms, interactions with the tumor microenvironment, and therapeutic implications. Pigment Cell Melanoma Res., 31, 11–30 (2018).
• 36) Kudo C, Yamakoshi H, Sato A, Nanjo H, Ohori H, Ishioka C, Iwabuchi Y, Shibata H. Synthesis of 86 species of 1,5-diaryl-3-oxo-1,4-pentadienes analogs of curcumin can yield a good lead in vivo. BMC Pharmacol., 11, 4 (2011).
• 37) Kudo C, Yamakoshi H, Sato A, Ohori H, Ishioka C, Iwabuchi Y, Shibata H. Novel curcumin analogs, GO-Y030 and GO-Y078, are multi-targeted agents with enhanced abilities for multiple myeloma. Anticancer Res., 31, 3719–3726 (2011).
• 38) Sugiyama S, Yoshino Y, Kuriyama S, Inoue M, Komine K, Otsuka K, Kohyama A, Yamakoshi H, Ishioka C, Tanaka M, Iwabuchi Y, Shibata H. A curcumin analog, GO-Y078, effectively inhibits angiogenesis through actin disorganization. Anticancer. Agents Med. Chem., 16, 633–647 (2016).
• 39) Shimazu K, Inoue M, Sugiyama S, Fukuda K, Yoshida T, Taguchi D, Uehara Y, Kuriyama S, Tanaka M, Miura M, Nanjyo H, Iwabuchi Y, Shibata H. Curcumin analog, GO-Y078, overcomes resistance to tumor angiogenesis inhibitors. Cancer Sci., 109, 3285–3293 (2018).
• 40) Bertazza L, Barollo S, Mari ME, Faccio I, Zorzan M, Redaelli M, Rubin B, Armanini D, Mian C, Pezzani R. Biological effects of EF24, a curcumin derivative, alone or combined with mitotane in adrenocortical tumor cell lines. Molecules, 24, 2202 (2019).
• 41) Huang X, Liang C, Yang H, Li X, Deng X, Liang X, Li L, Huang Z, Lu D, Ma Y, Luo Z. Curcumin induces apoptosis and inhibits the growth of adrenocortical carcinoma: identification of potential candidate genes and pathways by transcriptome analysis. Oncol. Lett., 21, 476 (2021).
• 42) Subramaniam D, May R, Sureban SM, Lee KB, George R, Kuppusamy P, Ramanujam RP, Hideg K, Dieckgraefe BK, Houchen CW, Anant S. Diphenyl difluoroketone: a curcumin derivative with potent in vivo anticancer activity. Cancer Res., 68, 1962–1969 (2008).
• 43) Liang Y, Yin D, Hou L, Zheng T, Wang J, Meng X, Lu Z, Song X, Pan S, Jiang H, Liu L. Diphenyl difluoroketone: a potent chemotherapy candidate for human hepatocellular carcinoma. PLoS ONE, 6, e23908 (2011).
• 44) Zhu S, Moore TW, Lin X, Morii N, Mancini A, Howard RB, Culver D, Arrendale RF, Reddy P, Evers TJ, Zhang H, Sica G, Chen ZG, Sun A, Fu H, Khuri FR, Shin DM, Snyder JP, Shoji M. Synthetic curcumin analog EF31 inhibits the growth of head and neck squamous cell carcinoma xenografts. Integr. Biol. (Camb.), 4, 633–640 (2012).
• 45) Bakalova R, Zhelev Z, Shibata S, Nikolova B, Aoki I, Higashi T. Impressive suppression of colon cancer growth by triple combination SN38/EF24/Melatonin: “Oncogenic” versus “Onco-Suppressive” reactive oxygen species. Anticancer Res., 37, 5449–5458 (2017).
• 46) Kasinski AL, Du Y, Thomas SL, Zhao J, Sun SY, Khuri FR, Wang CY, Shoji M, Sun A, Snyder JP, Liotta D, Fu H. Inhibition of IkappaB kinase-nuclear factor-kappaB signaling pathway by 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24), a novel monoketone analog of curcumin. Mol. Pharmacol., 74, 654–661 (2008).
• 47) Yang CH, Yue J, Sims M, Pfeffer LM. The curcumin analog EF24 targets NF-kappaB and miRNA-21, and has potent anticancer activity in vitro and in vivo. PLOS ONE, 8, e71130 (2013).
• 48) Selvendiran K, Tong L, Vishwanath S, Bratasz A, Trigg NJ, Kutala VK, Hideg K, Kuppusamy P. EF24 induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by increasing PTEN expression. J. Biol. Chem., 282, 28609–28618 (2007).
• 49) He G, Feng C, Vinothkumar R, Chen W, Dai X, Chen X, Ye Q, Qiu C, Zhou H, Wang Y, Liang G, Xie Y, Wu W. Curcumin analog EF24 induces apoptosis via ROS-dependent mitochondrial dysfunction in human colorectal cancer cells. Cancer Chemother. Pharmacol., 78, 1151–1161 (2016).
• 50) Zou P, Xia Y, Chen W, Chen X, Ying S, Feng Z, Chen T, Ye Q, Wang Z, Qiu C, Yang S, Liang G. EF24 induces ROS-mediated apoptosis via targeting thioredoxin reductase 1 in gastric cancer cells. Oncotarget, 7, 18050–18064 (2016).
• 51) Skoupa N, Dolezel P, Ruzickova E, Mlejnek P. Apoptosis induced by the curcumin analogue EF-24 is neither mediated by oxidative stress-related mechanisms nor affected by expression of main drug transporters ABCB1 and ABCG2 in human leukemia cells. Int. J. Mol. Sci., 18, 2289 (2017).
• 52) Zhang P, Bai H, Liu G, Wang H, Chen F, Zhang B, Zeng P, Wu C, Peng C, Huang C, Song Y, Song E. MicroRNA-33b, upregulated by EF24, a curcumin analog, suppresses the epithelial-to-mesenchymal transition (EMT) and migratory potential of melanoma cells by targeting HMGA2. Toxicol. Lett., 234, 151–161 (2015).
• 53) Olivera A, Moore TW, Hu F, Brown AP, Sun A, Liotta DC, Snyder JP, Yoon Y, Shim H, Marcus AI, Miller AH, Pace TW. Inhibition of the NF-kappaB signaling pathway by the curcumin analog, 3,5-bis(2-pyridinylmethylidene)-4-piperidone (EF31): anti-inflammatory and anti-cancer properties. Int. Immunopharmacol., 12, 368–377 (2012).
• 54) Nagaraju GP, Zhu S, Wen J, Farris AB, Adsay VN, Diaz R, Snyder JP, Mamoru S, El-Rayes BF. Novel synthetic curcumin analogues EF31 and UBS109 are potent DNA hypomethylating agents in pancreatic cancer. Cancer Lett., 341, 195–203 (2013).
• 55) Kusaba T, Nakayama T, Yamazumi K, Yakata Y, Yoshizaki A, Inoue K, Nagayasu T, Sekine I. Activation of STAT3 is a marker of poor prognosis in human colorectal cancer. Oncol. Rep., 15, 1445–1451 (2006).
• 56) Rajitha B, Nagaraju GP, Shaib WL, Alese OB, Snyder JP, Shoji M, Pattnaik S, Alam A, El-Rayes BF. Novel synthetic curcumin analogs as potent antiangiogenic agents in colorectal cancer. Mol. Carcinog., 56, 288–299 (2017).
• 57) Nagaraju GP, Zhu S, Ko JE, Ashritha N, Kandimalla R, Snyder JP, Shoji M, El-Rayes BF. Antiangiogenic effects of a novel synthetic curcumin analogue in pancreatic cancer. Cancer Lett., 357, 557–565 (2015).
• 58) Rajitha B, Belalcazar A, Nagaraju GP, Shaib WL, Snyder JP, Shoji M, Pattnaik S, Alam A, El-Rayes BF. Inhibition of NF-kappaB translocation by curcumin analogs induces G0/G1 arrest and downregulates thymidylate synthase in colorectal cancer. Cancer Lett., 373, 227–233 (2016).
• 59) Haque A, Rahman MA, Fuchs JR, Chen ZG, Khuri FR, Shin DM, Amin AR. FLLL12 induces apoptosis in lung cancer cells through a p53/p73-independent but death receptor 5-dependent pathway. Cancer Lett., 363, 166–175 (2015).