Effendi, et al.
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, 198-209
Co-Chemotherapy Effect of Glycosylated Nanoalbumin Genitri Seed Extract Targeting Induced Apoptotic on Overexpressed HER2 Breast Cancer Fatiha Citra Effendi1.
Intan Setyaningrum1.
Anindya Nasywa1.
Eliana Bella1.
Ratna Asmah Susidarti2,3* Bachelor of Pharmacy Study Program.
Faculty of Pharmacy.
Gadjah Mada University.
Indonesia Department of Pharmaceutical Chemistry.
Faculty of Pharmacy.
Gadjah Mada University.
Indonesia Cancer Chemoprevention Research Center.
Faculty of Pharmacy.
Universitas Gadjah Mada Abstract HER2-targeted therapy aims to stop proliferation and induced apoptosis.
Genitri seeds show anticancer effects and have potential as co-chemotherapy.
To maximize bioactive delivery, a glycosylated-nanoalbumin delivery system is used.
This research aims to explore the ability of Glycosylated-Nanoalbumin Genitri Seeds (GN-GSE) to induce apoptosis in MCF-7/HER2 cancer cells.
The cytotoxic test using the MTT assay showed GSE selectivity (SI=2.
against cancer cells (IC50 104 g/mL) and non-toxicity against normal cells (IC50 284 g/mL).
Induction of apoptosis occurs through inhibition of the CDK1 protein which is predicted by molecular docking.
GSE has the potential as a synergistic co-chemotherapy with tamoxifen (CI<.
and has good affinity for the CDK1 inhibitory domain.
Elaeocarpenine is known to have good affinity with a greater iG value (-11.
40 kcal/mo.
than the native The GN-GSE formula meets the criteria for nanoparticles with good stability.
This research shows that GSE has anti-cancer activity, making it potential as a therapy for HER2 overexpressed breast cancer as well as in a glycosylated nanoalbumin drug delivery system.
Keywords: Apoptotic induced, co-chemotherapy, genitri seeds, glycosylated, nanoalbumin.
INTRODUCTION
In 2020, breast cancer was the most prevalent cancer in Indonesia, affecting 30.
8% of cancer patients, with nearly 20% of these cases HER2
(GCO,
Martinez-Saez and Prat, 2.
HER2
overexpressed breast cancer is a specific subtype characterized by the overexpression of the human epidermal growth factor receptor-2 (HER2 ), which contributes to more rapid growth and metastase compared to other types of breast cancer (Novitasari, et al.
, 2.
Unfortunately, inadequate treatment of this malignancy can lead to increased severity, recurrence, and even mortality.
Therefore, targeted therapies focusing on HER2 are Submitted: October 14, 2024 Revised: May 27, 2025 Accepted: May 28, 2025 *Corresponding author: ratna_asmah@ugm.
Indonesian Journal of Cancer Chemoprevention.
October 2024 ISSN: 2088Ae0197 e-ISSN: 2355-8989 a valuable approach, as they play an anti-apoptotic role by activating ERK, which regulates apoptosis (Amtiria and Berawi, 2.
Consequently, inducing apoptosis is a crucial goal in the treatment of HER2 overexpressed breast cancer.
To achieve better clinical outcomes in cancer therapy, combination of herbal treatments become favorable, driven by the side effects and complications of long-term drug therapies like This approach, known as co-chemotherapy, allows for reduced doses while minimizing side effects (Zulfin, et al.
, 2.
One notable local wisdom from Malanesia is genitri (Elaeocarpus ganitru.
, often called the "King of Herbal Medicine" for its analgesic, antibacterial, antifungal, and anticancer properties (Mahajanakatti, et al.
The alkaloid compound Elaeocarpenine in genitri seeds contributes to its anticancer effects (Primiani, et al.
, 2.
Furthermore, genitri seed extract exhibits cytotoxic effects on PC-3 cancer cells, which can be enhanced by nanoparticle delivery system (Vinay, et al.
, 2.
The selection of a cancer drug delivery system considers both aspects of selectivity and bioavailability, particularly for non-polar compounds like Elaeocarpenine .
ogP value 3.
considered as lipophilic compound.
(Vinay, et al.
PubChem, 2.
Therefore, glycosylated drug delivery nanoalbumin formula was chosen because it enhances both properties.
Glycosylated drug delivery employs glycosylation as a delivery vehicle to target GLUT receptors on the cell Cancer cells often overexpress glucose receptors to obtain energy for metabolism and growth, allowing selective drugs to target cancer cells more effectively than normal cells (Costa, et , 2.
Nanoalbumin acts as a bioactive carrier to improve cellular entry and bioavailability (Li, et al.
, 2.
This study aims to determine if Glycosylated Nanoalbumin from Genitri Seeds (GNGSE) can enhance cytotoxicity against HER2 overexpressing breast cancer, supporting its role as an apoptosis-inducing co-chemotherapy agent.
MATERIALS AND METHODS
Location and Time of Research The research was conducted for 4 months in the Pharmaceutical Laboratory and Pharmaceutical Chemistry Laboratory of the Faculty of Pharmacy, as well as the Parasitology Laboratory of the Faculty of Medicine.
Public Health, and Nursing (FKKMK) UGM.
Tools and Materials The tools used include magnetic stirrer (Scientifi.
Buchner flask (Iwak.
, rotary shaker (Stuar.
, 1-20 AAL micropipette (Gilson.
Middleton.
WI.
USA), 100-1000 AAL micropipette (Gilso.
, 96-well (Cornin.
MOE 2010 software, ultraturrax (IKA T.
Brookfield
(LAMY
Reolog.
, pH-meter (Hann.
, and Particle Size Analyzer (Malver.
The materials used include genitri seeds (Kebumen.
Central Java.
Indonesi.
BSA (Sigma-Aldrich.
St.
Louis.
Missour.
PierceTM (Thermo-scientific.
Waltham.
MA.
USA), aquadest (Wateron.
, methanol p.
a (Merck.
Darmstadt.
German.
, silica plate 60 (Sigma-Aldric.
Dragendorff reagent (Merc.
, anisaldehyde-H2SO4 (Merc.
, citroborate (Merc.
, ethyl acetate (Merc.
, (Merc.
, (Merc.
, dichloromethane (Merc.
MTT reagent (SigmaAldric.
Tamoxifen (Sigma-Aldric.
MCF7/ HER2 cell-lines (ATCC).
Vero cell-lines (ATCC).
DMEM-high glucose (Gibc.
, fetal bovine serum (Gibc.
Penicillin Streptomycin (Gibc.
Trypsin 0.
25% (Gibc.
, sodium dodecyl sulfate (Sigma-Aldric.
Method of Collecting data Genitri Extraction and Phytochemical Profiling Identification The genitri seed extraction method was adapted from Primiani, et al.
, which employed maceration techniques using methanol Effendi, et al.
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Cancer Chemoprevent.
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a for 24 h .
olid: liquid 1:.
to obtain GSE.
The solvent evaporated with rotary evaporator.
Phytochemical profiling with Thin Layer Chromatography (TLC) used a mobile phase of hexane: ethyl acetate: acetic acid .
%:25%:20%).
The major alkaloid compound, elaeocarpenine, found in GSE was detected by the Dragendorff reagent (Gowthama, et al.
, 2.
Cytotoxicity Test with MTT Assay Cells were seeded in 96-well plates at a density of 5x103 cells/well.
5 mg of GSE was dissolved in 50 AAL of DMSO.
Tamoxifen was prepared in three concentration series: 0.
05 AAM,
1 AAM, and 0.
2 AAM.
The sample solutions were added to the wells and incubated for 24 h, after which MTT reagent was treated.
After formazan was formed, a 10% SDS solution was added to stop the reaction, and the absorbance was measured using an ELISA reader at a wavelength of 595 The IC50 value was calculated based on linear regression of the percentage of cell viability against the GSE concentrations (Ikawati, et al.
, 2.
Molecular Docking with MOE Molecular docking was performed to explore the apoptosis mechanism and signaling proteins involved.
The target protein (CDK.
file complexed with native ligand was downloaded from PDB .
ttps://w.
org/).
The docking process was performed with the default MOE Compounds were subjected to structural visualization with output in the form of iG .
ocking scor.
indicating the strength of the ligand-receptor interaction (Lestari and Utomo.
Glycosylated-Nanoalbumin Genitri Seed Extract (GN-GSE) Formulation Albumin (BSA) was conjugated with fructose in a 1X PBS buffer .
mL) at a ratio of 2:3 and stirred at 200 rpm.
The mixture was then freeze-dried, following the method described by Asyhari, et al.
To prepare GN-GSE, the freeze-dried samples were oven-dried at 50AC for durations of 60, 90, and 120 minutes.
The resulting samples were dissolved in NaCl at pH 7.
Table 1.
Glycosylated albumin and GN-GSE preparation formulation.
Material Weight .
Albumin (BSA) Fructose PBS 1x
400 mg
600 mg
5000 mg
*Freeze-dried for 1x24 h and heated in the oven at 50AC for 60, 90, and 120 minutes Glycosylatedalbumin
GSE
NaCl PBS 1x Additionally.
GSE was dissolved in ethanol and mixed, after which the solution was freeze-dried, as noted by Huang, et al.
Evaluation of Albumin Glycosylation The glycosylation index evaluation was conducted using the Bradford test.
A dilution series of non-glycosylated BSA was prepared, 4 mg 2 mg 2 mL Ad to 5 mL ranging from 1000Ae200 AAg/mL, using distilled The GN-GSE samples were weighed and subsequently dissolved in distilled water.
A total of 10 AAL of the BSA dilution series and the samples were placed into a 96-well plate.
Additionally, 150 AAL of Pierce reagent was added and incubated for 15 minutes.
The absorbance was then measured with a plate reader .
, and the amounts of Indonesian Journal of Cancer Chemoprevention.
October 2024 ISSN: 2088Ae0197 e-ISSN: 2355-8989 glycosylation albumin was calculated.
Furthermore, the quality parameters of the preparation included particle size analysis (PSA), viscosity testing, and pH stability assessment.
RESULTS
Genitri Seed Extraction (GSE) Genitri seed samples (Elaeocarpus ganitru.
, family Elaeocarpaceae, were obtained from a genitri plant cultivation house in Kebumen.
Extraction was carried out using the ultrasonicassisted extraction (UAE) method at 40AC for 45 This method implies efficient extraction of alkaloid.
The duration was considered enough to exerts the pores within hard-shield seeds (AguilarHernyndez, et al.
, 2.
A total of 2 g of genitri seeds were extracted with 20 mL of methanol solvent .
olid:liquid 1:.
, the macerate solvent was evaporated and the crude extract obtained was 110 mg with yield of 5.
5% .
The extract obtained was fractionated with dichloromethane.
After evaporation of the solvent, the fraction weight obtained was 25 mg.
Phytochemical Profiling Genitri Seed Extract (GSE) Using Thin Layer Chromatography (TLC) A total of 5 AAL of GSE-crude extract (C.
and GSE-fractioned extract (F.
,000 pp.
was spotted on a silica 60 plate and eluted with a mobile phase of hexane: ethyl acetate: acetic acid .
%: 25%: 20%).
The elution results were then dipped in Dragendroff's reagent to detect alkaloid compounds, sitroboric reagent to detect flavonoid compounds, and Anisaldehyde-H2SO4 reagent to detect terpenoid compounds.
After heating, alkaloid compounds were marked with brown spots with Rf 46 in visible light.
Flavonoid compounds were marked with fluorescent spots with Rf 0.
73 in UV366.
Meanwhile, terpenoid compounds are marked with spots with Rf 0.
57 in visible light.
Figure 1.
TLC profile of GSE after the addition of dragendorff reagent indicated the amount of alkaloid compounds .
Sitroboric reagent reagent indicated the amount of flavonoid compounds .
and Anisaldehyde AeH2SO4 indicated the amount of terpenoid compounds .
Cytotoxic Effects of GSE on MCF-7/HER2 and Vero Cells Cytotoxic effects indicate the toxicity of GSE .
ractioned extrac.
to cells based on the value IC50.
MCF-7/HER2 cells represent cancer cells and Vero cells represent normal cells.
After GSE treatment, it was found that there was a decrease in cell density and changes in cell The viability profile of MCF7/HER2 cells due to GSE administration showed an IC50 of Effendi, et al.
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Figure 2.
Cytotoxic effect of GSE on MCF-7/HER2 cancer cell line and Vero normal cell line.
Cell viability profile of MCF-7/HER2 , the obtained IC50 104 AAg/mL (R=0.
Microscopy morphological of MCF-7/HER2 treated GSE .
Cell viability profile of Vero, the obtained IC50 284 AAg/ mL (R=0.
Microscopy morphological of Vero treated GSE .
104 AAg/mL.
Meanwhile, the viability profile of Vero cells due to GSE administration showed an IC50 of 284 AAg/mL.
The selectivity index (SI) obtained revealed the value of 2.
7, indicating that GSE was selective for cancer cells.
assessed by measuring free amino acid levels using the Bradford Protein Assay.
Results showed that after 120 minutes, the free amino acid level decreased to 29.
29%, indicating a significant extent of glycosylation.
GlycosylatedAeAlbumin Formulation The Glycosylated-BSA complex formation relies on the Maillard reaction, a non-enzymatic process where free amino groups in proteins interact with reducing sugars to create cross-link In this study, bovine serum albumin (BSA) and fructose were combined and dissolved in phosphate-buffered saline (PBS), followed by vortexing and freeze-drying.
The resulting freeze-dried samples were then subjected to heating at 50AC for varying durations of 60, 90, and 120 The success of glycosylation was Glycosylated-Nanoalbumin Genitri Seed Extract (GN-GSE) Formulation and Preparation Quality Control Test GN-GSE formulation via dispersion BSA and fructose were freeze-dried to increase nanoparticle stability and increase the shelf-life of the formulation (Fonte, 2.
The preparation has physical properties of clear yellow, odorless, and a viscosity value of 10 cPs indicating that this formula is in the medium viscosity range (Berteau, et al.
, 2.
The stability of the preparation for 3 weeks at 2AC storage showed a pH stability of 7.
Indonesian Journal of Cancer Chemoprevention.
October 2024 ISSN: 2088Ae0197 e-ISSN: 2355-8989 Evaluation of the preparation particles was carried out to confirm the characteristics of the nanoparticles.
Based on the PSA characterization.
GN-GSE has a particle size of 75.
46 nm according to the size of Nano sized of 10-1000 nm becomes one of factors that influences effectiveness of drug GN-GSE preparation has met the requirements of a homogeneous nano preparation with a PdI value of 0.
42 (PdI<0.
(Veronika.
Figure 3.
Glycosylation Index of BSA.
Standard curve of BSA concentrations obtained by regression, y=0.
The level of free amino acids after heating process represents the amount of albumin glycosylation index.
Cytotoxic Combination Effects Glycosylated-Nanoalbumin Genitri Seed Extract (GN-GSE) and Chemotherapy Drugs GN-GSE formulation was obtained by glycosylation of BSA and fructose, which entrapped GSE, and act as its carrier.
A cytotoxicity combination was conducted to evaluate the effects of GN-GSE on the cytotoxic effects of tamoxifen in MCF7/HER2 cells.
The results suggest that a synergistic combination of GN-GSE and tamoxifen can enhance the efficacy of tamoxifen, enabling a reduction in the treatment dose while still achieving the same level of cytotoxic effect (Muna and Jenie.
Specifically, a combination of 100 AAg/mL of GN-GSE with varying concentrations of tamoxifen showed a synergistic effect, indicated by a Figure 4.
Cytotoxic combination of GN-GSE and Tamoxifen.
Cell viability profile of MCF-7/HER2 treated GN-GSE .
ontaining GSE 50.
200 g/mL) and Tamoxifen .
7 M).
Combination Index (CI) value of GSEAeTamoxifen.
CI <1.
0 determined as synergistic combination.
Effendi, et al.
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Cancer Chemoprevent.
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Combination Index <1.
0 (Haagensen, et al.
, 2.
Overall, the combination of GN-GSE with tamoxifen was found to inhibit cell growth more effectively than either treatment alone at the same Molecular Docking of Apoptosis Regulator Proteins With MOE 2010 The affinity interaction of GSE compoundsinduced apoptosis was conducted through molecular docking on the CDK1 target protein.
Target protein selection was based on data from the UALCAN .
ttps://ualcan.
edu/analysis.
, which identifies overexpressed genes in HER2 breast cancer, and Swiss Target Prediction .
ttp://swisstargetprediction.
ch/), which indicates proteins targeted by specific compounds.
Results were evaluated using InteractiVenn to analyze overlaps (Chandrashekar, et al.
, 2.
CDK1 (CyclinDependent Kinase .
regulates the G2/M phase of the cell cycle.
The docking results indicated that the main GSE compounds had a stronger affinity for CDK1 than the native ligand.
Dinaciclib, a known A more negative docking score .
G) signifies better affinity and inhibition.
Consequently, three GSE compounds were identified as effective CDK1 inhibitors, as shown in Table 2 and Figure 5.
Ligand Tabel 2.
S-score docking senyawa utama GSE.
Native ligand* Elaeocarpenine Elaeocarpucine C S-score 80 kcal/mol 40 kcal/mol 71 kcal/mol Quercetin 92 kcal/mol Figure 5.
Visualization of ligand interaction on CDK1.
The native ligand shown in blue-colored, while the target compounds shown in orange-colored: A.
Elaeocarpenine.
Quercetin.
Elaeocarpucine C.
DISCUSSION
This research aims to explore the cytotoxic effects of Genitri Seed Extract (GSE) as a co-chemotherapy agent that induces apoptosis in HER2-overexpressing breast cancer formulated in glycosylated drug delivery nanoalbumin.
Genitri plants (Elaeocarpus ganitru.
are known to have anticancer effects with moderate cytotoxicity against BxPC-3 and HeLa cell lines (Utami, et al.
, 2.
The anticancer activity of genitri plants is supported by flavonoid, terpenoid, and alkaloid compounds, with a typical compound, namely elaeocarpenine, which is found in large quantities in the seeds Indonesian Journal of Cancer Chemoprevention.
October 2024 ISSN: 2088Ae0197 e-ISSN: 2355-8989 (Yogesh, et al.
, 2.
Extraction of compounds carried out with methanol to dissolve polar and nonpolar analytes in GSE, one of which is Alkaloid fractionation is carried out by dichloromethane due to its volatile nature and the presence of chlorine groups can facilitates the separation of compounds and protects compounds from damage due to high temperatures (Emilda, et , 2.
Phytochemical profiling of GSE was performed using TLC with visualization reagents to identify the presence of compounds.
Dragendorff's reagent detected alkaloids, yielding a reddish-orange color (Chairunnisa, et al.
, 2.
The Sitroboric reagent was used to identify flavonoids, it reacts with the ortho-hydroxy group yielding a bright yellow color that fluorescence under UV254 visualization (Hikmawanti, 2.
, while anisaldehyde-H2SO4 revealed terpenoids with a purple color (Masadi, et al.
, 2.
The analysis found alkaloids at Rf of 0.
47, flavonoids at Rf 0.
and terpenoids at Rf 0.
57, which linked to anticancer The anticancer effects of GSE were studied using the MCF-7/HER2 breast cancer cell model, which overexpresses the HER2 receptor.
This receptor activates proliferation and antiapoptotic signals through the PI3K pathway.
Bcl-2, and IAP proteins, inhibiting apoptosis (Siddiqa, et , 2.
The safety of GSE was also tested on Vero cells, an African green monkey kidney epithelial The IC50 value for MCF-7/HER2 cells was 104 g/mL, while for Vero cells, it was 284 g/mL, yielding a selectivity index (SI) of 2.
7, indicating a preference for cancer cells over normal cells (Artun et al.
, 2.
Combination of GSE with the chemotherapy agent, tamoxifen, showed a synergistic effect with CI value of less than 1.
0 at most concentrations (Ho, et al.
, 2.
This synergy may enhance therapy effectiveness, reduce drug resistance, and lower side effects (Ho, et al.
, 2.
HER2 mutations in HER2 type breast cancer cause chemotherapy resistance and increased dimerization of the HER2 family (Ron, et al.
, 2.
The application of glycosylated drug delivery nanoalbumin helps overcome the mechanism of cancer Figure 6.
Albumin glycosylation proposed mechanism.
drug resistance by specifically targeting cancer cells utilizing the principle of glycosylation of glucose derivatives of aerobic glycolysis as a characteristic of cancer cells (Koppenol, et al.
, 2.
Protein nanoparticles, offer various benefits compared Biocompatibility, biodegradability, and low toxicity are the characteristics of protein nanocarriers (Verma.
Glycation is based on the Maillard reaction, which is the process of conjugating proteins with reducing sugars through covalent bonds upon heating which forms glycated protein products (Asyhari, 2.
Glycated success parameters based on the Bradford Protein Assay.
The principle of this method involves binding protein molecules with coomassie blue in acidic conditions which causes a color change from brown to blue.
A decrease in albumin concentration correlates with a decrease in % free amino which indicates that the -amino group of tripeptide has been bound to the carbonyl group of sugar (Suseno, 2.
The best result obtained the lowest free amino of 29.
29% at a Effendi, et al.
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temperature of 50AC for 120 minutes.
The appropriate heating duration can catalyze the Maillard reaction which faster the glycosylation process (Asyhari, 2.
According to Primiani, et al.
the methanolic extract of GSE contains unique phytochemical compounds from the Elaeocarpus family, including 1.
88% Elaeocarpenine, 1.
Elaeocarpucine C, and high amount of Quercetin These compounds represent the major constituents of alkaloids, terpenoids, and flavonoids.
The presence of these 3 classes of compounds was confirmed by TLC, indicating that Elaeocarpenine.
Elaeocarpucine C, and Quercetin were present as it were the major components.
GN-GSE selectively accumulates in cancer cells through glucose transporters overexpressed on the cell membrane.
GLUT-5 is responsible for fructose uptake and is overexpressed in MCF-7 cell line in the breast cancer model (Zhou, et al.
, 2.
In MCF-7/HER2 cells, increased proliferation occurs due to the binding of the CDK1 complex to cyclin B1 in the G2-M phase.
HER2 dimerization activates the PI3K pathway and ERK pathways which causes a decrease in p21 expression as a CDK1 inhibitor.
The decrease in CDK1 inhibitors makes the activation of the proliferation pathway uninhibited (Saatci, et al.
, 2.
In this case, the affinity of the GSE compound complex as a CDK1 inhibitor is an important target for inducing Figure 7.
Proposed mechanism of apoptosis induction by disruption of cyclin-CDK1 complex.
The bioinformatics study using molecular docking demonstrated that these three major compounds from GSE exhibit strong binding affinity to CDK1, with the following values:
Quercetin at -13.
92 kcal/mol.
Elaeocarpucine C 71 kcal/mol.
Elaeocarpenine at -11.
40 kcal/ mol in comparison with native ligand Dinaciclib at 80 kcal/mol.
Although Elaeocarpenine was the unique compound of GSE.
Quercetin has higher affinity among the rest of two major compounds, followed by Elaeocarpucine C, than Elaeocarpenine.
The presence of these 3 major compounds suggests that GSE may alter the interaction between CDK1 and cyclin B way better, thereby preventing its activation and influencing cell cycle arrest on G2/ M-phase.
The more negative iG value, the stronger the binding affinity and the more effective on inhibition of CDK1 (Ortiz, et al.
, 2.
The binding of Elaeocarpenine.
Elaeocarpucine C, and Quercetin to CDK1 prevents CDK1 from Indonesian Journal of Cancer Chemoprevention.
October 2024 ISSN: 2088Ae0197 e-ISSN: 2355-8989 associating with cyclin B1, leading to cell cycle This mechanism is what induces apoptosis in breast cancer cells (Wijnen, et al.
, 2.
CONCLUSION
GSE contains alkaloids, flavonoids, and terpenoids that exhibit anticancer properties.
GSE
specifically targets cancer cells, such as MCF-7/ HER2 , while being non-toxic to normal cells like Vero.
The apoptosis-induced effect by GSE occurs through the inhibition of CDK1, a mechanism that shows a strong affinity, as indicated by a higher s-score value of the target compound compared to the native ligand.
Additionally, the glycosylated drug delivery formulation of GN-GSE demonstrates good stability over a storage period of three weeks and meets the criteria for nanoparticles.
ACKNOWLEDGMENT
We express our gratitude to Ministry of Research.
Technology and Higher Education.
Republic of Indonesia.
PKM Program 2024, and Cancer Chemoprevention Research Center (CCRC) for funding and supporting this research.
REFERENCES