Muthi Ikawati

Breast cancer stem cells (bCSCs) exhibit high plasticity, therapeutic resistance and immune evasion, making them critical targets for effective cancer immunotherapy. Pentagamavunone-1 (PGV-1), a curcumin analog, and its derivative, Chemoprevention Curcumin Analog-1.1 (CCA-1.1), possess cytotoxic and reactive oxygen species (ROS)-modulating properties. However, their potential role in modulating immune-related phenotypes in bCSCs remains unclear. In the study, bCSCs derived from the MDA-MB-231 cell line were treated with PGV-1 or CCA-1.1 to evaluate cytotoxicity, apoptosis, cell cycle distribution, stemness-associated markers and immune checkpoint expression. Cell viability was assessed using the CCK-8 assay. Apoptotic, stemness and immune-checkpoint profiles were analyzed using flow cytometry. PGV-1 exhibited stronger cytotoxicity against bCSCs than CCA-1.1, characterized by lower IC50 values (10.70 and 28.78 µM for PGV-1 and CCA-1.1 respectively at 24 h) and greater induction of apoptosis in a dose-dependent manner. Both compounds at concentrations of 2.67 and 7.19 µM significantly (p < 0.001) induced G2/M cell cycle arrest and reduced the CD44+CD24– stem-like population, indicating loss of stemness and enhanced differentiation. Importantly, PGV-1 significantly downregulated PD-L1 while upregulating CD80, suggesting a shift toward immunophenotypic alterations associated with a potentially more immunogenic profile. These dual effects of cytotoxic and preliminary immunophenotypic modulation highlight the ability of PGV-1 to overcome immune resistance in bCSCs. Further investigation using immune cell–based functional assays or in vivo models is required to validate whether the observed immunophenotypic modulation translates into functional immune activation and to assess the potential of PGV-1 in a co-immunotherapy setting.

Pentagamavunon-1 (PGV-1) is a potential anticancer candidate due to its specific targeting of cancer cells with minimal toxicity to normal cells. This study examined the potency of hesperidin (HSD) in improving the anticancer effects of PGV-1 for triple-negative breast cancer (TNBC). The 4T1 and NIH-3T3 cell lines were used as the model of TNBC and normal fibroblast, respectively. The cytotoxicity was quantified using MTT and clonogenic assays, whereas the cell cycle and apoptosis were measured by flow cytometry. Cell migration was assessed by scratch-wound healing assay. Senescence was measured using the SA-β-gal assay, and the secretion of matrix metalloproteinase (MMP)-2 and 9 was examined using gelatin zymography. Protein expression level was analyzed through capillary Western blot. The collected data were quantified using one-way ANOVA. PGV-1 significantly reduced 4T1 cell viability, with IC ₅₀ values of 7 and 2 µM at 24 and 48 h, respectively. Its cytotoxic effect persisted up to 7 days after removal. HSD alone showed no cytotoxicity against 4T1 cells, but in combination with 1 µM of PGV-1, it synergistically enhanced cell growth inhibition and apoptosis induction. This synergism is likely due to ERK1/2 phosphorylation inhibition, leading to reduced levels of Bcl-2 and elevated levels of Bax. In NIH-3T3 cells, higher concentration PGV-1 (10 µM) induced early senescence-associated features, but 100 µM HSD prevented this and suppressed MMP-2 and 9 secretion. PGV-1 and HSD combination demonstrates potential as a co-treatment strategy for effective anticancer therapy while preserving normal cell integrity.

Doxorubicin has been incorporated in cancer therapy regimes for wide-range malignancies, but it causes many side effects. These adverse effects can be overcome by combining doxorubicin with other ingredients that possess anticancer potential but are safe on normal cells. The tubers of Merremia mammosa, locally known as “Bidara Upas” in Indonesia, have been proven to have anticancer potential with resin glycosides as the active compounds. This research aims to develop the extract of M. mammosa’s tubers (MTE) as a co-chemotherapy agent for doxorubicin. A malignant cancer cell line, 4T1, was used as the model. MTE was obtained by maceration in 96% ethanol. The thin layer chromatography confirmed that MTE contains glycoside compounds. Administration of MTE to 4T1 cells showed cytotoxic activity with an IC50 value of 61 μg/mL as evaluated by MTT assay. The combination of MTE and doxorubicin exhibited synergistic cytotoxic effects with a combination index of <0.7. Moreover, MTE at around IC50 was able to cause DNA fragmentation indicating apoptosis as observed by the agarose gel electrophoresis.These data support our hypothesis that MTE may serve as a potential co-chemotherapeutic agent for doxorubicin; however, the apoptosis-inducing potency of the combination requires further investigation.Keywords: 4T1 cell, apoptosis, Bidara Upas, co-chemotherapy.

The immune system plays an important role for the body, especially in protecting it from exposure to bacteria, viruses, and other foreign bodies. Improving the immune system can be done by daily consumption of certain foods. Foods that can be developed into an immunostimulant are tubers, one of which comes from the genus Dioscorea. Dioscorea esculenta L., known as gembili tuber, is widely found in Indonesia, but has not been widely tested for its activity as an immunostimulant both against the innate and adaptive immune systems. This study aims to determine the immunostimulant activity of aqueous extract (AE), polysaccharide fraction (PF), and non-polysaccharide fraction (NPF) of gembili tubers against macrophage phagocytosis activity and lymphocyte proliferation. Test of phagocytosis activity and lymphocyte proliferation was performed in vitro by adding AE, PF, NPF gembili tubers 12.5, 50, and 100 mg/mL, inulin 100 µg/mL, and positive control 10 mg/mL as a comparison in macrophage cells and mouse lymphocyte cells. Phagocytosis activity was expressed in phagocytosis capacity and phagocytosis index, while lymphocyte proliferative activity was expressed in proliferative stimulating index. The results showed that AE, PF, and NPF could increase macrophage phagocytosis activity, with the highest activity observed at AE 100 μg/ml, PF 100 μg/ml, and NPF 12.5 μg/ml. AE, PF, and NPF were also shown to increase lymphocyte proliferation activity, with the most significant enhancement observed at AE 12.5 μg/mL, PF 12.5, and NPF 50 μg/mL.

N-Myc and c-Myc, as two major members of the MYC family proteins, independently contribute to hepatocellular carcinoma (HCC) by promoting proliferation, survival, and metabolic reprogramming. Overexpression of both proteins correlates with poor clinical outcomes, while intrinsic structural disorder hinder direct pharmacological targeting of MYC proteins. CCA-1.1 exhibits enhanced biological and physicochemical properties over its parent compound, curcumin, and demonstrates potent anti-cancer activity in various cancer types. We aim to investigate the anti-proliferative activity of CCA-1.1 through modulation of the MYC proteins and induction of cell cycle arrest and/or cell death mechanisms, including senescence, apoptosis, and autophagy. CCA-1.1 exhibited potent and irreversible anti-proliferative activity on Huh-6 cells by inhibiting EGFR kinase activity and its downstream signalling. CCA-1.1 also promoted G2/M arrest, induced apoptosis, and generated senescence. At the molecular level, CCA-1.1 downregulated N-Myc and c-Myc protein levels through the promotion of GSK3β activation and suppression of NCYM, a stabilizer protein of N-Myc. Furthermore, CCA-1.1 activated autophagy as a result of MYC protein destabilization. CCA-1.1 exerts a multi-faceted anti-cancer effect in MYCN-expressed HCC cells by inhibiting kinase signalling and destabilizing MYC proteins, leading to apoptosis, senescence, and autophagy. Molecular docking and molecular dynamic simulation revealed that CCA-1.1 interacted with both N-Myc and c-Myc. Our findings demonstrate CCA-1.1 as a distinctive anti-cancer candidate for HCC driven by MYC activation.

Cancer is the most common cause of death, with the burden increasing in recent years. Conventional treatments have caused cancer cells’ resistance to drugs. Therefore, it is crucial to develop a radio-labeled theranostic compound such as penta-gamma-vunone (PGV), which significantly suppresses cancer cell growth. In this experiment, the synthesis of [131I]I-PGV-0 compound was done by using the standard Chloramine-T (CT) iodination method with some parameter optimizations, such as the quantity of CT oxidizer and sodium metabisulfite reducing agent, time and temperature of reaction. The radiochemical purity of these compounds was determined using thin layer chromatography (TLC) analysis, and the stability of the radiolabeled compound was also assessed in HBSS medium. Results showed that the highest radiochemical purity (RCP) of [131I]I-PGV-0 (99.83%) was obtained at the CT amount of 25 µg. Stability study revealed that, at the high RCP, this compound was stable for 16 days in cold storage. Molecular docking studies indicated that the radioiodination of PGV-0 enhances binding affinity by interacting with crucial amino acids within the kinase domains of EGFR, HER2, AURKA, and PLK-1.

Context: Breast cancer remains the second leading cause of cancer-related mortality worldwide, driven by uncontrolled cellular proliferation and metastasis. Natural radiopharmaceuticals have gained interest due to their potential for reduced side effects compared to synthetic agents. Among these, hesperidin and hesperetin, especially in iodinated forms, have emerged as promising therapeutic candidates targeting key breast cancer-related signaling pathways. Aims: To evaluate iodinated hesperidin derivatives as multi-target breast cancer therapeutics using molecular docking (AutoDock) and dynamics (GROMACS). Methods: Multi-target molecular docking and 100 ns molecular dynamics simulations were conducted to examine interactions with key breast cancer proteins: EGFR kinase, human estrogen receptor alpha (ER-α), and I-kappa-B-alpha/NF-kappa-B. Binding energies (∆G) were computed, and complex stability was assessed using RMSD, RMSF, and MM-PBSA analyses. Pharmacokinetic and toxicity properties were also predicted. Results: Docking results indicated strong affinities for iodinated derivatives (-6.14 to -7.51 kcal/mol), notably with EGFR and ER-α. Molecular dynamics simulations confirmed enhanced stability and reduced structural fluctuations in iodinated complexes. MM-PBSA calculations showed improved binding free energies, supporting increased interaction strength. Despite lower solubility, ADME predictions suggested favorable gastrointestinal absorption and drug-likeness for iodinated hesperetin. Toxicity analyses predicted moderate nephrotoxicity and cardiotoxicity risks, with lower LD50 values, indicating a need for dose optimization. Conclusions: Iodinated hesperidin and hesperetin exhibit stable binding interactions and favorable pharmacological profiles, highlighting their potential as novel radiopharmaceuticals in breast cancer therapy. These findings warrant further preclinical investigation and optimization for future drug development.

Ursolic acid, a triterpenoid compound found in various plants, has been garnered significant attention as potential co-chemotherapeutic agent for breast cancer therapy. To comprehensively evaluate the current state of research and identify knowledge gaps, we conducted a bibliometric analysis of the literature pertaining ursolic acid and breast cancer. Using several databases, including Scopus, Google Scholar, and PubMed, we analyzed publication trends, collaboration patterns, and research foci within this field from 2000 to 2024. The highest number of publications was from China, which had 37 documents on those three databases. The most cited article was published by Yeh et al. in 2010. The keyword co-occurrence analysis discovered that “cytotoxic effect”, “breast cancer cell line”, and “therapy” were terms that are most frequently used in this field. Our bioinformatic study revealed that ursolic acid has ability to bind into MAPK3 protein with affinity score -7.2 kcal/mol due to van der Waals and alkyl interactions involving several amino acid residues. This negative regulation of MAPK3 can inhibit cell proliferation, metastasis, and glucose metabolism in breast cancer cells. Overall, this bibliometric analysis and bioinformatic study provide valuable insights into the current landscape of ursolic acid research for breast cancer therapy and highlights areas that warrant further investigation.

Purpose: SARS-CoV-2 infection may lead to a worse prognosis in COVID-19 patients by inducing syncytia formation which implies intercellular transmission and immune evasion. Hesperidin (HSD) and hesperetin (HST) are two citrus flavonoids that demonstrate the potential to interfere with spike/hACE2 binding and show an inhibitory effect in the SARS-CoV-2 pseudovirus internalization model. Here, we determined the effects of HSD and HST to inhibit syncytia formation using in vitro cell models. Methods: We confirmed spike, hACE2, and TMPRSS2 ectopic expressions by immunofluorescence staining (IF) after transfection using polyethylene imine (PEI) in 293T cells. Then, the cells were transfected with a set of plasmids encoding spike/hACE2/TMPRSS2 or spike/hACE2 to induce syncytia formation. Cell treatment with HSD/HST was performed 4-5 h after transfection and then incubated for another 16-18 h. Syncytia were observed using an inverted microscope or a high content screening (HCS) platform. The data obtained from syncytia formation assays were statistically analyzed using ANOVA (Bonferroni). Results: We successfully observed spike, hACE2, and TMPRSS2 expression in 293T cells by IF staining. Furthermore, we showed that HSD 10 and 100 µM significantly inhibited the formation of small-to-medium-sized syncytia compared to the control cells by manual syncytia observation. In the HCS assay, 10 µM HSD showed an inhibitory effect of syncytia induced by spike WT. In contrast, 100 µM HSD, 10 and 100 µM hesperetin (HST), and 10 µg/mL citrus peel extract containing HSD prepared by the hydrodynamic cavitation method (HCV) inhibited syncytia formation induced by spike Omicron. Conclusion: HSD and HST show the potential inhibitory activity of SARS-CoV-2 intercellular transmission. Further study is needed to confirm the mechanism of action of the antiviral activity.

Purpose: The HLF and HuH-6 cell lines represent hepatocellular carcinoma (HCC) with different characteristics in chromosome content that may give different drug responses. Here, PGV-1 was intended to challenge the growth-suppressing effect on HLF and HuH-6 and trace the molecular target mechanism of action compared to sorafenib. Methods: We applied MTT cytotoxic assay, colony forming assay, flow cytometry analysis, immunofluorescence assay and western blot assay. Results: PGV-1 exhibited cytotoxic effects on HLF and HuH-6 with IC-50 values of 1 µM and 2 µM, respectively, whereas sorafenib showed less cytotoxicity with IC-50 values of 5 µM and 8 µM respectively. PGV-1 suppressed the cell growth permanently but not for sorafenib. Sorafenib did not change the cell cycle profiles on both cells, but PGV-1 arrested the cells at G2/M with the characteristic of senescent cells and mitotic disarrangement. PGV-1 and sorafenib showed the same effect in downregulating p-EGFR, indicating that both compounds have the same target on EGFR activation or as Tyrosine kinase inhibitors. PGV-1 suppressed the MYCN expression in HuH-6 and HLF cells but stabilized cMYC-T58 indicating that even though the MYCN was downregulated, the cells maintained the active form of cMYC. In this regard, PGV-1 also stabilized the expression of PLK-1 and AurA. Conclusion: PGV-1 elicits stronger cytotoxic properties compared to sorafenib. The lower the MYCN expression, the higher the cytotoxic effect of PGV-1. PGV-1 abrogates cell cycle progression of both cells in mitosis through EGFR inhibition and stabilizes PLK-1 and AurA in correlation with the suppression of MYCN expression.