The potency of Pentagamavunone‐0 (PGV‐0) as chemopreventive agent for the formation and growth of breast cancer as revealed in 3D model

Pentagamavunone‐0 (PGV‐0) or 2,5‐bis(4’‐hydroxy‐3‐methoxybenzylidine)‐cyclopentanone is a curcumin analogue that exhibits anticancer activity in breast cancer cells. However, most of previous reports are limited to the use of two‐dimensional (2D) cell culture. The use of three‐dimensional (3D) cell culture model in cancer research can represent the real condition of cancer growth in patients better than the 2D culture. The purpose of this study was to determine the anticancer activity of PGV‐0 on a 3D model of HCC 1954 breast cancer cells. HCC 1954 cells were grown in the 3D culture in the presence of PGV‐0, and the spheroid formation and growth of formed spheroids were observed using microscope at 24 and 96 h, respectively. The cytotoxic effects were measured by MTT assay. PGV‐0 inhibited the formation and growth of spheroids at the concentration as low as 60 μM. The cytotoxic effect of PGV‐0 appeared in a dose‐dependent manner with the IC50 value of 70.9 μM. The results of this study indicate that PGV‐0 has an anticancer activity on a 3D model of HCC 1954 breast cancer cell line. Therefore, the result supported the potency of PGV‐0 as cancer chemopreventive agent.


Introduction
Chemoprevention is the process of preventing or inhibit ing the development of cancer by using chemopreven tive agents, either natural or synthetic chemical sub stances (Meiyanto et al. 2012). Natural compounds are widely used as chemopreventive agents because of their safety, low toxicity, antioxidants, and used as food sup plements. Curcumin, epigallocatechin 3gallate (EGCG), resveratrol, sulforaphane, and withaferinA are natural compounds that have potential as chemopreventive agents (Pistollato et al. 2017). Pentagamavunone0 (PGV0) or 2,5bis(4'hydroxy 3methoxybenzylidine)cyclopentanone is an analogue of curcumin. Synthesis of PGV0 was carried out to increase the solubility level of curcumin in water and maintain its stability at pH>6.5 (Mohammadian et al. 2019; Utomo et al. 2017. It was reported to have similar or better anti inflammatory activities than curcumin. PGV0 can inhibit the growth of T47D breast cancer cells through apoptosis and has antiproliferative effects on myeloma cells (Da'i et al. 2004(Da'i et al. , 2007. PGV0 also shows cytotoxic activity in WiDr colon cancer cells (Septisetyani et al. 2008) and MCF7 breast cancer cells (Hermawan et al. 2011). The previously reported anticancer research of PGV0 is lim ited to the use of twodimensional (2D) cell culture and had not been carried out on threedimensional (3D) cell culture.
The 3D cell culture performs better abilities to repre sent the real condition of cancer cells in the patients com pared to the 2D culture. These better abilities include cell morphology, cellcell communication, gene expression, and biological and stress response (Boutin et al. 2018). In 2D cell cultures, cancer cells attach and grow on a flat sur face resulting one layer of proliferating cells; while can cer cells in 3D cultures form aggregates or spheroids, 3D shaped cancer cells, consisting of proliferation, quiescent, and necrotic zones (Edmondson et al. 2014). Concern ing the stress response, the 3D model is also suitable to explore the resistance characteristic or the stem celllike phenomenon.
The physiological mechanism of PGV0 as a candi date for chemopreventive agent needs to be explored fur ther, mainly focusing on the stress response. Curcumin has been widely explored for its cytotoxic activities to many types of cancer cells. The high selectivity of curcumin to cancer cells rather than normal cells possibly a correlates with the inhibitory activities against some reactive oxy gen species (ROS) metabolizing enzymes that contribute to the induction of cell apoptosis as well as cell migra tion (Larasati et al. 2018). Therefore, PGV0 may have the same mechanism as curcumin that triggers cell death in a 3D system. This research was carried out to deter mine the biological activity of PGV0 as a candidate for chemopreventive agents for breast cancer by using HCC 1954 breast cancer cells in a 3D model.

Materials
Curcumin and PGV0 in powder forms were obtained from Cancer Chemoprevention Research Center (CCRC), Fac ulty of Pharmacy, Universitas Gadjah Mada, Indonesia. Curcumin (271.5 mM) and PGV0 (283.8 mM) stock solu tions were prepared by dissolving them in dimethyl sulfox ide (DMSO) (Sigma Aldrich). The stock solutions were then dissolved in culture medium at a serial concentration 10, 20, 30, 60, 80, and 160 µM to treat the cancer cells.

Spheroid formation
The formation of spheroids was carried out by growing HCC 1954 cells (9 × 10 3 cells/well) in the RPMI medium on 96well plates that had been coated with 1.5% agarose matrix (Sigma). The cells were incubated for 96 h until the spheroids were formed and ready to be used for exper iments (Bashari et al. 2019). The morphological appear ances of the spheroids were observed by using an inverted microscope (Boeco) at 24, 48, 72, and 96 h. The diame ter of the spheroids was measured using Image Raster 3.0 software while the density of the spheroids was measured using ImageJ software.

Inhibitory effect of spheroid formation
To find out the inhibitory effect of PGV0 and curcumin on spheroid formations, HCC 1954 cells (9 × 10 3 cells/well) were grown in RPMI medium on an agarosecoated 96 well plate. The cells then were treated with 100 µL/well of PGV0 or curcumin at a serial concentration (0, 10, 20, 30, 60, 80, 100, and 160 µM). Spheroids were formed after 24 h incubation.

Inhibitory effect of spheroid growth
The effect of PGV0 and curcumin on the growth of spheroids can be observed after the spheroids had been formed. The spheroids were treated with PGV0 or cur cumin at a serial concentration and incubated for 96 h.

Cytotoxicity assay
The cytotoxic effect of PGV0 and curcumin were mea sured using MTT assay according to the previous report (Ho et al. 2012) with a slight modification to the proto col. Briefly, the spheroids which had been treated with PGV0 or curcumin for 96 h were then transferred to a new 96well plate using a micropipette. Ten micro liters of MTT reagents were added to each wellcontaining spheroid in 90 µL of the transferred medium. The reaction was stopped by adding an SDS stopper solution containing 0.01 N HCl to the spheroids that had been incubated for 4 h. Furthermore, the spheroids were incubated overnight at room temperature in a dark place. Absorbance was mea sured using an ELISA reader with a wavelength of 595 nm. Percentage of the cell viability was calculated to determine IC 50 values by a linear regression analysis ).

Spheroid formation
The spheroids of HCC 1954 breast cancer cells began to form at 24 h, but the compactness was relatively low. The compactness increased as the incubation time increased. It caused a decrease in the diameter of spheroids during the incubation period ( Figure 1B). The highest reduction of spheroids diameter occurred from 24 h to 48 h, which was 7%, while the total decrease in spheroid diameter during the incubation period was 12% ( Figure 1C). The cohesive ness of spheroids was obtained by measuring the spheroid density based on the color of the observed images. The color of spheroids was getting darker during the incuba tion period ( Figure 1A), which means that the thickness of the spheroids was increasing ( Figure 1D).

PGV0 inhibits spheroid formation
Spheroids were formed after incubation for 24 h ( Figure  2A). PGV0 began to inhibit the formation of spheroids at 60 µM. It could be seen that at this concentration, HCC 1954 cells could not form compact aggregates. Some HCC 1954 cells partially scattered at the edge of noncompact aggregates as a single cell ( Figure 2B). Under 60 µM, PGV0 resulted in spheroids with the cohesiveness de crease as the increase of the concentrations. Curcumin could inhibit the formation of spheroids at a lower concen tration than PGV0, which is 30 µM. At this concentration, HCC 1954 cells formed smaller compact aggregates than untreated spheroids and were surrounded by noncompact aggregates. The density of the spheroids was measured using ImageJ software. Each point on B and D was measured from three wells and is presented as mean ± standard deviation; while point on C was calculated based on the data from B and is represented in average. The asterisk (*) indicates a significant difference (P<0.05).

PGV0 inhibits spheroid growth
The data showed that PGV0 and curcumin caused decreases and increases, respectively, in the formed spheroids ( Figure 3A). PGV0 increased the spheroid di ameter at a concentration of 60 μM while curcumin at a concentration of 30 μM, but not at the lower concentration ( Figure 3B). After 96 h, at the lower concentration, the hi hgest total reduction in spheroid diameter was 4% by 30 μM PGV0; while 30 μM curcumin caused the highest in crease as many as 8% ( Figure 3C).

PGV0 has cytotoxic activity on spheroids
The cytotoxic activity of PGV0 and curcumin in spheroids was measured by MTT assay. PGV0 and curcumin per formed a cytotoxic effect on a dosedependent manner in HCC 1954 breast cancer cells with the IC 50 of 70.9 µM and 60.6 µM, respectively (Figure 4).

Discussion
This study aimed to determine the anticancer activity of PGV0 on a 3D model of HCC 1954 breast cancer cells by inhibiting the formation, growth, and viability of spheroids. Therefore, it was started by growing the spheroids without being treated to find out the period of spheroids to be formed, the incubation period of spheroids, and the spheroid appearance during the incubation period. The result showed that spheroids began to be formed at 24 h with the compactness increased as the incubation period increased. It was indicated by the decreasing diameter and increasing density of spheroids during 96 h of incubation. Spheroids were ready to use after being incubated for 96 h in accordance to the previous study (Bashari et al. 2019). The spheroids are ready to use when there is a decrease in diameter, but the size of the spheroids is relatively the same.
The use of 2D culture in cancer research has limita tions as it consists of one layer of cells so that it cannot rep resent the real condition of cells in patients which consists of several layers of cells. The 3D cell culture has a better ability to represent cell morphology, cellcell communica tion, gene expression, and biological response compared to 2D culture (Boutin et al. 2018). In this study, PGV0 has tested on 3D models of HCC 1954 cells. Besides PGV0, curcumin was also used in this study as a comparison.
The results indicated that PGV0 could inhibit the for mation of spheroids, starting at a concentration of 60 µM. The inhibitory effect on spheroids increased as the concen tration increased. Curcumin treatment at a serial concen tration (0160 µM) started to inhibit at a concentration of 30 µM. Although curcumin was able to inhibit at a lower concentration than PGV0, the aggregates coming from PGV0 treatment were more fragile by pipetting. It may indicate that PGV0 had a better ability to reduce adhesion between cancer cells than curcumin. Epithelialcadherin (Ecadherin) is a transmembrane glycoprotein that has a pivotal role in maintaining cell to cell adhesion (Liu and Chu 2014). The inhibitory effect on spheroids formation might be caused of PGV0 and curcumin effects on the E cadherin expression. However, the molecular mechanism of this phenomenon needs to be explored further.
PGV0 showed the ability to inhibit the growth of spheroids by decreasing and increasing the diameter. The spheroid diameter started to increase at a concentration of 60 µM. Furthermore, the inhibitory effect of spheroid growths had not been seen under a concentration of 60 µM. This can be occurred because the spheroids were ex periencing the same condition as untreated spheroids that were decreasing in diameter. Besides, the cohesiveness of spheroid increased, and only cells in the outer part of the spheroids began to detach. In contrast, an increase of the spheroid diameter might be caused by a decrease in adhe sion between cells due to PGV0 so that the cells detach in all parts of the spheroids. The reduced sticking abil ity between cells resulted in the decreased spheroid cohe siveness. This result also happened to the treatment with curcumin, but at a lower concentration of 30 µM.
The previous study reported that a single treatment of PGV0 exhibited anticancer activities in several types of 2D breast cancer cells, namely T47D (Da'i et al. 2007), MCF7 (Hermawan et al. 2011), and MCF7/HER2 (Meiyanto et al. 2014). In combination with other chemotherapy agents, PGV0 enhances their cytotoxic ef fects in a 2D model of MCF7 and WiDr cells (Hermawan et al. 2011; Ikawati andSeptisetyani 2018) so that it can be served the candidate of a cochemotherapy agent. This study showed that the treatment of PGV0 exhibits cyto toxic effects on spheroids of HCC 1954 breast cancer cells with the IC 50 value of 70.9 µM. This value was about twice the IC 50 of PGV0 in 2D models of HCC 1954 cells (CCRC unpublished data, 2019). PGV0 affects spheroids with a higher concentration than monolayer cells because the spheroids consist of several layers so that the toxic effects are not evenly distributed on all cells. The outermost part of spheroids obtained greater influence than the inner part (Sant and Johnston 2017). The toxic effects increase as the concentration increases meaning that the higher the con centration, the fewer the number of living cells. Mean while, the treatment of curcumin at the same serial con centration (0100 mΜ) gave an IC 50 value of 60.6 μM. The results showed that curcumin has a higher cytotoxic effect on a 3D model of HCC 1954 cells than PGV0. Taken to gether, PGV0 and curcumin have a cytotoxic activity at an intermediate level in the range of 21200 µM.
In this study, curcumin performs a better effect than PGV0 as it affects the spheroids at a lower concentration. Anticancer activity of PGV0 is not always better than cur cumin in all types of cancer cells. In WiDr colon cancer cells, curcumin had a higher cytotoxic effect than PGV0 with an IC 50 value of 27 μM; while PGV0 is 45 μM (Sep tisetyani et al. 2008). Curcumin has been well known to have anticancer activity (Meiyanto 1999). It is proven to inhibit the growth of cancer cells by inducing apoptosis (Da'i et al. 2017) and increasing ROS levels beyond the threshold (Larasati et al. 2018). Therefore, PGV0 may inhibit the growth of cancer cells in the same pathway as curcumin. PGV0 still has an opportunity to be used as a candidate of chemopreventive agent for breast can cer treatment because the active concentration is similar to curcumin but has more stable and better bioavailabil ity than curcumin. Nevertheless, further investigation is needed to reveal the molecular mechanism of cells death caused by PGV0.

Conclusions
PGV0 performed the anticancer activity in the 3D model of HCC 1954 breast cancer cell line. It was seen from the ability of PGV0 to inhibit the formation and growth of spheroids and its toxic effect. Based on the results, PGV0 can be promoted as a candidate of chemopreventive agent for breast cancer treatment.