: Pentagamavunon-1 performs more potent anti-cancer effects than curcumin against various cancer cells, but it remains to be optimized. Piperine shows the activity as an enhancer of a therapeutic agent. This study expects to achieve higher effectiveness of PGV-1 on 4T1 breast cancer cells through co-treatment with piperine with exploring the effect of cytotoxicity, mitotic catastrophe, cellular senescence, and target proteins of PGV-1 and piperine on the regulation of mitosis in TNBC cells (4T1). The assays emphasize MTT assay, May Grünwald-Giemsa staining, SA-β-galactosidase assay, and bioinformatics analysis, respectively, to elicit the respected activities. The results revealed that PGV-1 performed a cytotoxic effect with an IC50 value of 9 µM while piperine showed a lower cytotoxic effect with an IC50 value of 800 µM on 4T1 cells 24 h treatment. However, the combination treatment of both showed a synergistic cytotoxic enhancement effect with an average CI value < 1. Furthermore, the combination of PGV-1 and piperine induced mitotic catastrophe and senescence better than the single treatment. Treatment of 1 µM of PGV-1 and 400 µM of piperine increased the percentage of senescent cells by 33%. Bioinformatics analysis revealed that PGV-1 and piperine target proteins play a role in mitotic regulation, namely CDK1, KIF11, AURKA, AURKB, and PLK1, to contribute to mitotic catastrophe. Therefore, piperine increases the effectiveness of PGV-1 to suppress 4T1 cells growth synergistically that may occur through mitotic catastrophe and senescence targeting on mitotic regulatory proteins.

Objective: To evaluate the anti-cancer properties of Caesalpinia sappan and Ficus septica in combination with doxorubicin on 4T1 cells, confirm their nephroprotective activities, and predict the molecular targets of the underlying mechanisms. Methods: The cytotoxic activities of all extracts and doxorubicin were determined by MTT assay followed by cell cycle and apoptosis analysis using flow cytometry. Immunoblotting was used to determine the protein expressions. The proteins involved in the cell proliferation and migration were analyzed through bioinformatics approaches, whereas, the interaction between compounds and protein targets was observed through molecular docking. Furthermore, the effect of the extracts on cell migration was analyzed by scratch wound healing assay. The intracellular ROS after treatment with extracts was observed using DCFDA staining flow cytometry. Results: Both ECS and EFS performed cytotoxic properties and significantly enhanced doxorubicin's cytotoxic effects against 4T1 cells. However, these cytotoxic activities did not correlate with the cell cycle progression. On the contrary, the combination treatment caused apoptosis that may correlate with the decreasing of IκBα phosphorylation, indicating that all agents targeted the inhibition of NF-κB activation. The combination treatments also inhibited cell migration and decreased MMP-9 expression. TNBC proliferation and metastasis needed at least 54 proteins to be activated, some of them are related to NF-κB activation. The inhibitory effect of ECS correlated with the interaction of brazilin and brazilein to IKK, a kinase protein that plays a role in IκBα phosphorylation. In addition, ECS and EFS reduced ROS expression in Vero cells caused by doxorubicin. Conclusion: In conclusion, ECS and EFS effectively enhanced the cytotoxic effect of doxorubicin and inhibit cell migration on 4T1 cells and these activities may correlate to the inhibitory effect of NF-κB activation. ECS and EFS also exhibit ROS suppressing effect on Vero cells that may be beneficent to reduce nephrotoxicity of chemotherapeutic treatment.

Pentagamavunon-1 (PGV-1), a potential chemopreventive agent with a strong cytotoxic effect, modulates prometaphase arrest. Improvement to get higher effectiveness of PGV-1 is a new challenge. A previous study reported that the natural compound, galangin, has antiproliferative activity against cancer cells with a lower cytotoxicity effect. This study aims to develop a combinatorial treatment of PGV-1 and galangin as an anticancer agent with higher effectiveness than a single agent. In this study, 4T1, a TNBC model cell, was treated with a combination of PGV-1 and galangin. As a result, PGV-1 and galangin showed a cytotoxic effect with IC50 values of 8 and 120 µM, respectively. Combining those chemicals has a synergistic impact, as shown by the combination index (CI) value of 1. Staining with the May Grunwald-Giemsa reagent indicated mitotic catastrophe evidence, characterized by micronuclear and multinucleated morphology. Moreover, the senescence percentage was higher than the single treatment. Furthermore, bioinformatics investigations showed that PGV-1 and galangin target CDK1, PLK1, and AURKB, overexpression proteins in TNBC that are essential in regulating cell cycle arrest. In conclusion, the combination of PGV-1 and galangin exhibit a synergistic effect and potential to be a chemotherapeutic drug by the mechanism of mitotic catastrophe and senescence induction.

Background We previously reported that in highly metastatic breast cancer cells, doxorubicin (DOX) at non-toxic concentrations promoted cell migration and invasion. Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavonoid glycoside isolated from citrus/lemon plant that possesses a cytotoxic effect in several cancer cells. In this study, we investigate whether DOX efficacy is enhanced by hesperidin (Hsd) and the molecular pathway involved in highly metastatic breast cancer, 4T1. Methods Combined cytotoxicity of Hsd and DOX was evaluated with MTT assay and was analyzed using Chou-Talalay’s method. To better understand the underlying mechanism, several factors, including apoptosis and cell cycle arrest were analyzed by flow cytometry. In addition, antimigration activity was evaluated by scratch wound healing assay, MMP-9 expression by ELISA and gelatin zymography, and Rac-1 protein level using western blot. The data on survival rate and expression level of MMP-9 and Rac-1 were obtained from Gene Expression OMNIBUS (GEO). Results Under MTT assay, Hsd showed a cytotoxic effect in a concentration-dependent manner with an IC50 value of 284 µM on 4T1 cells. Hsd synergistically enhanced the cytotoxic effect of DOX which seemed to correlate with an increase in apoptotic cell death, G2/M cell cycle arrest and blocked the migration of 4T1 cells. At 10 nM, doxorubicin induced lamellipodia formation, and increased the level of Rac-1 and metalloproteinase-9 (MMP-9) expression. Interestingly, combined treatment of DOX and Hsd dramatically downregulated the expression of MMP-9 and Rac-1. These results indicated that Hsd block the cell migration induced by DOX under in vitro studies. Conclusion These findings strongly suggest that Hsd possesses a potential synergistic effect that can be developed to enhance the anticancer efficacy of DOX and reduce the risks of chemotherapy use in highly metastatic breast cancer.

Objective: This study aimed to determine the cytoprotective potentials of citronella ( Cymbopogon nardus (L.) Rendl.) essential oil (CO) and lemongrass ( Cymbopogon citratus (DC.) Stapf) essential oil (LO). Methods: The essential oils from citronella and lemongrass were obtained by steam-water distillation, then analyzed using Gas Chromatography-Mass Spectrophotometry (GC-MS) to determine the chemical constituents. The antioxidant activity of CO and LO was compared using a total antioxidant capacity kit. The viability of normal kidney epithelial cells Vero and fibroblast NIH-3T3 as the cell models were tested using a trypan blue exclusion assay. The effect of cellular senescence inhibition on both cell models was measured using senescence-associated β-galactosidase (SA-β-gal) staining. The mechanism of action of CO and LO in the protection of cellular damage against doxorubicin was also confirmed through 2’,7’–dichlorofluorescin diacetate (DCFDA) staining to discover the ability to decrease reactive oxygen species (ROS) levels and a gelatin zymography assay to observe the activity of matrix metalloproteinases (MMPs). Results: The major marker components of CO and LO were citronellal and citral, respectively. Both oils showed low cytotoxic activity against Vero and NIH-3T3 cells, with IC 50 values of over 40 µg/mL. LO exhibited higher antioxidant capacity than CO, but there was no effect on the intracellular ROS level of both oils on Vero and NIH-3T3 cells. However, CO and LO decreased cellular senescence induced by doxorubicin exposure on both cells, as well as suppressed MMP-2 expression. Conclusion: Both CO and LO decrease the cellular senescence and MMP-2 expression with less cytotoxic effects on normal cells independently from their antioxidant capacities. The results were expected to support the use of CO and LO as tissue protective and anti-aging agents in maintaining the body’s cellular health against chemotherapeutics or cellular damaging agents.