INTRODUCTION:

Curcumin is a natural polyphenol compound derived from the plant Curcumin longa¹. It has shown to inhibit cancer cell proliferation through cell cycle induction²^,³, improve apoptosis⁴ and inhibition of metastasis⁵^,⁶. Curcumin is a potential compound to be developed as a chemotherapy and chemopreventive agent⁷. However, the presence of an active methylene group causes curcumin to be less stable to light⁸, besides that low solubility is still a significant problem in the development of curcumin⁸^,⁹^,¹⁰. In addition, PGV-1 is a curcumin analog with the mono-ketone structure that has anti-cancer activity through cell cycle arrest and increases apoptosis in T47D and MCF 7 breast cancer cells¹¹^,¹².

Breast cancer is one of the highest causes of death in the world¹³. More than one million women are diagnosed with breast cancer, and more than 410,000 women die from breast cancer each year¹⁴^,¹⁵. Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor-2 (Her2) and consider as the most malignant type of breast cancer¹⁶^,¹⁴.

Although associated with a generally poor breast cancer-specific outcome, most TNBC is not resistant to chemotherapy¹⁷. Therefore, several therapies are being developed to target specific biomarkers of TNBC subtype, including EGFR-targeted agents, androgen receptor-targeted agents, anti-antigenic agents, and PARP¹⁸. However, more work is needed to identify targets that yield high therapeutic ratios¹⁹^,²⁰. Recently, a promising therapeutic target for TNBC includes the Wnt/b-Catenin signaling pathways²¹.

Metastasis is a complex biological process that remains to be a significant problem in the management of cancer²². Mechanism of metastasis includes separation of the primary tumor, migration, and survival in the circulation system²³^,²⁴. This process is regulated by several proteins, such as β-catenin and GSK-β. β-catenin would bind to cadherin and will affect actin cytoskeleton, which will provide cancer cells migration²¹^,²⁵. In this research, β-catenin was used as a protein target in the process of metastatic cancer cells. The purpose of this study was to analyze the effects of curcumin and PGV-1 on migration 4T1 breast cancer and in silico study analyze of the molecular interactions of curcumin and PGV-1 with β-catenin and GSK-3β.

MATERIALS AND METHODS:

Chemicals:

Curcumin (Sigma), PGV-1 has been synthesized by Curcumin Research Center (CRC), Faculty of Pharmacy, Universitas Gadjah Mada.

Cell culture:

The 4T1 cells were obtained from ATCC. The cells were cultured in RPMI medium supplemented with L-Glutamine, 10% v/v FBS (Gibco), 1, 5% v/v penicillin and streptomycin, and maintained in a CO₂incubator at 37^o C.

Wound-healing assay:

The evaluation of cell migration was conducted by wound-healing assay. 4T1 cells were seeded at 7.5x10⁴ cells/well in 6-well plates and incubated for 24 h at 37°C. Cultured cells were washed with PBS and added culture media, which containing 10% FBS and incubated for 24 h. Scratch was done in the bottom center of the well within the cell layer using a yellow tip. Residues cell in the plate were washed with PBS and treated with curcumin, or PGV-1, and incubated for 48 h at 37°C and documented under the inverted microscope against cell migration rapidity after 0, 24, and 48 h. The space from scratch treatment between control and treated cells was quantified using Image-J software and defined as cell migration area.

Molecular Docking: Molecular docking analysis was conducted by evaluating the molecular interactions of curcumin and PGV-1 against β-catenin (PDB: 4DJS) and GSK-3β (PDB: 4ACH) proteins. The structure of curcumin and PGV-1 were taken from Pubchem. These experiments were perform using Molecular Operating Environment (MOE) 2015 (licenses from faculty of pharmacy UGM). Validation process refers to literature26. The molecular docking protocol used the alpha triangle placement algorithm and London dG scoring function.

RESULTS AND DISCUSSION:

Curcumin or diferuloylmethane [1, 7-bis-(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-dione], a yellow pigment, is a major component of turmeric and a member of the ginger Curcuma longa⁸. The potential for anti-cancer curcumin through its activity in suppressing the proliferation of various tumor cells; regulate β-catenin and migration cell in HEK293 cell²⁷. PGV-1 (Fig. 1) is a curcumin analog which has anti-cancer activity. The previous study, it was showed that the IC50 values of curcumin and PGV-1 were 34.34ug/ml and 38.21 ug/ml, respectively, in 4T1 breast cancer cells.

Curcumin and It is Analogue Inhibit 4T1 cell migration

Migration ability is one of the hallmarks of tumor metastasis²⁸. To further examine the possible anti-migration ability of 4T1 breast cancer cell, a wound-healing assay was performed. Image of scratch areas from 0, 24, and 48 h are illustrated in Fig.3. As shown in Fig. 3a, treatment of 4T1 breast cancer cell with ½ IC50 dose of curcumin and PGV-1 separately for 24 h inhibited the cell migration respectively by 20,67% and 2,95% compared to non-treated control cell, whereas, the inhibition of migration in 48 h respectively by 25,47% and 3,65% compared to control. The results clearly showed that treatment with curcumin and PGV-1 caused a significant inhibition of cell migration in the 4T1 breast cancer cell. Hence, this result show PGV-1 more potent to inhibit cell migration than curcumin in 4T1 breast cancer. A recent study report that curcumin could inhibit colon cancer cell migration²⁹^,³⁰ and melanoma skin cancer³¹. Moreover, Kim and Mukherjee team show the mechanism of anti-migration of curcumin through inhibition of β-catenin and GSK-3β in Hep3B hepatocarcinoma and breast cancer cell³²^,³³.

	0 h	24 h	48 h
Control
Curcumin
PGV-1

(A)

(B)

Figure 2. Curcumin and PGV-1 inhibit breast cancer cell migration: (A) Cell migration assay in 4T1 breast cancer cells. (B) Graph showing quantification of rate migration. *P< 0.05, (mean ± SEM, n = 3)

Molecular Interaction of Curcumin and Its Analogue through β-catenin and GSK-3β

β-catenin is considered to be an essential protein engaged in the initiation and progression of metastatic breast cancer³⁴. In the canonical model of Wnt signaling, β-catenin is phosphorylated at specific key residues by glycogen synthase kinase-3b (GSK-3β) leading to its ubiquitination and subsequent degradation. Translocation of β-catenin into the nucleus promotes migration via the destruction of E-cadherin complexes and action myosin mechanism³⁵-³⁶.

Table 1. Molecular interaction of curcumin and PGV-1 against β-catenin and GSK-3β

	β-catenin			GSK-3β
	Residue	Interaction	Scoring	Residue	Interaction	Scoring
Curcumin	Asn516	Pi bond	- 10,9717	Phe67	π-π interaction	- 12,2149
				Asp 90	Hydrogen bond
				Glu97	Hydrogen bond
PGV-1	His470	Pi bond	-13,3036	Asp200	Hydrogen bond	- 13,5285
	Lys435	Hydrogen bond
	Asn430	Hydrogen bond

This research uses molecular docking studies to determine the interaction of curcumin and PGV-1 against the active sites of β-catenin and GSK-3β using MOE software (version 2015). This study revealed that curcumin binds with both β-catenin and GSK-3β with a considerably high binding energy. Curcumin binds into the active site of β-catenin (4DJS) and GSK-3β (4ACH). The docking results also confirmed that curcumin strongly binds with an amino acid residue of β-catenin at Asn516, while PGV-1 bind at His470, Asn430, and Lys435. Furthermore, curcumin and PGV-1 bind site active of GSK-3β at Phe67, Asp 90 and Glu97, whereas PGV-1 interact at Asp 200. These amino acid residues actively participate in hydrophobic, hydrophilic, and π-π interactions. Several recent studies show that curcumin suppresses β-catenin and GSK-3β in Wnt pathway³². Curcumin inhibits migration and proliferation of medulloblastoma cell by suppressing signaling of β-catenin³⁷. Curcumin and PGV-1 inhibit cell migration through binding with the active site of β-catenin and GSK-3β. The above mechanism need to be explored further.

The present study shows curcumin analog is more potent than curcumin to inhibit metastasis. The use of curcumin and PGV-1 decrease cancer metastasis through inhibit cell migration. Inhibition of β-catenin and GSK-3β involve with decreasing cell migration. This makes PGV-1 a potential therapeutic agent for triple negative breast cancer in future.

CONCLUSION:

Our study show curcumin and its analog reduce cell migration of 4T1 breast cancer. Computational modeling show curcumin and PGV-1 interact with the active site of β-catenin and GSK-3β. This result indicated that PGV-1 has the potency to be developed as chemotherapeutic through inhibition of migration by interacting with β-catenin and GSK-3β.

ACKNOWLEDGMENT:

The authors would like to thank Ritmaleni for PGV-1 synthesis. This work was supported by Ministry of Research Technology and Higher Education through the research scheme of PBK year 2017-2018 with contract number of 1729/UN1/DITLIT/DIT-LIT/LT/2018 for funding this research.

AUTHORS CONTRIBUTION:

All of the authors listed in the manuscript has contributed equally.

CONFLICT OF INTEREST:

The authors declare there is no conflict of interest is associated with this work.

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Received on 10.09.2019 Modified on 24.10.2019

Research J. Pharm. and Tech. 2020; 13(4):1715-1719.

DOI: 10.5958/0974-360X.2020.00309.1