The Cytotoxic and Anti-Migratory Properties of Caesalpinia sappan and Ficus septica, in Combination with Doxorubicin on 4T1 TNBC Cells with Nephroprotective Potential.

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.

Different Cytotoxic Effects of Vetiver Oil on Three Types of Cancer Cells, Mainly Targeting CNR2 on TNBC.

OBJECTIVE: To investigate vetiver oil (VO) selectivity effects on several cancer cell types and identify the ß-caryophyllene role and mechanisms to prevent cancer development. METHODS: Cytotoxic effects of VO on three types of cancer cells (WiDr, 4T1, T47D) were determined using MTT assay. VO's effects on the cell cycle and apoptosis were analyzed using flow cytometry. Intracellular Reactive Oxygen Species (ROS) of cells after treatment with VO was observed with DCFDA staining. Bioinformatics study and molecular docking were used to determine the molecular targets of VO. RESULTS: VO contained various essential oils in which ß-caryophyllene was the most abundant. 4T1 cells performed the lowest IC50 value. WiDr and 4T1 cells showed an arrest in the G2/M phase, while T47D showed an increase of sub G1 population after VO treatment. On the other hand, apoptosis was only observed in WiDr and T47D cells. ROS levels were increased significantly in WiDr and T47D cells but not in 4T1 cells. Cannabinoids CB2 receptor (CNR2) was highly expressed in 4T1 cells and commonly exhibited a low survival rate on Triple Negative Breast Cancer (TNBC) patients. CNR2 was the notable target of ß-caryophyllene and performed agonistic interaction, which might have contributed to its cytotoxic activity against 4T1 cells. CONCLUSION: The molecular interaction of VO cannabinoid agonists and the CNR2 receptor was the underlying cause of VO cytotoxicity, which is a VO distinction on TNBC. Therefore, VO is better suited for use as an anti-cancer agent in TNBC cells.

Synergistic Cotreatment Potential of Soursop (Annona muricata L.) Leaves Extract with Doxorubicin on 4T1 Cells with Antisenescence and Anti-reactive-oxygen-species Properties.

Annona muricata L. extract (AME) exhibits cytotoxic activities on various types of cancer cells. This study aims to unveil the anticancer activity of AME as a cotreatment agent with doxorubicin (dox) on 4T1 cells and AME's relation to senescence. AME was obtained by maceration using 96% ethanol. AME was then subjected to qualitative analysis using TLC compared to quercetin (hRf = 75). Spectrophotometry analysis of AME resulted in a total flavonoid content of 2.3% ± 0.05%. Cytotoxic evaluation using the MTT assay revealed that AME showed an IC50 value of 63 µg/mL, while its combination (25 µg/mL) with dox (10 nM) decreased the viability of 4T1 cells to 58 % (CI = 0.15). Flowcytometry using propidium iodide staining confirmed that AME (13 and 25 µg/mL) caused cell cycle arrest in the G1 phase as a single treatment and G2/M arrest in combination with dox. However, by using the dichloro dihydrofluorescein diacetate staining assay, it turned out that AME at concentrations of 13 and 25 µg/mL decreased intracellular reactive oxygen species (ROS) levels both as a single treatment and in combination with dox. Senescence-associated ß - galactosidase assay showed that AME decreased dox-induced senescence. AME alone and in combination with dox (cotreatment) showed cytotoxic effect synergistically on 4T1 cells, but this was not caused by an increase in intracellular ROS levels as well as senescence induction. Therefore, AME showed its potential to be a cotreatment agent with antioxidant property on triple-negative breast cancer cells.

Reactive oxygen species and senescence modulatory effects of rice bran extract on 4T1 and NIH-3T3 cells co-treatment with doxorubicin

Objective:To determine the effect of rice bran extract (RBE) in combination with doxorubicin on 4T1 triple-negative breast cancer cells and NIH-3T3 cells. Methods:RBE was obtained by maceration with n-hexane. The phytochemical profile of RBE was observed using high-performance liquid chromatography. Cytotoxic effect of RBE was evaluated through MTT assay. In addition, flow cytometry was used for cell cycle and apoptosis analysis. Cellular senescence was observed using SA-β-Gal assay and intracellular reactive oxygen species (ROS) levels were evaluated using DCFDA staining. The pro-oxidant property of RBE was also evaluated through 1-chloro-2,4-dinitrobenzene spectrophotometry and molecular docking. Results:RBE was obtained with a yield of 18.42％ w/w and contained tocotrienols as the major compound. RBE exerted no cytotoxic effect on 4T1 and NIH-3T3 cells. However, RBE in combination with doxorubicin decreased 4T1 cell viability synergistically (combination index<0.9) and induced apoptosis and senescence on 4T1 cells. RBE significantly decreased senescence in doxorubicin-treated NIH-3T3 cells. Additionally, RBE did not increase ROS levels in doxorubicin-treated 4T1 cells. Meanwhile, the combination of RBE and doxorubicin reduced ROS levels in NIH-3T3 cells. RBE significantly reduced glutathione-S-transferase activity and alpha-tocotrienol interacted with glutathione-S-transferase in the glutathione binding site. Conclusions:Rice bran may be used as a co-chemotherapeutic agent to improve the therapeutic effectiveness of doxorubicin while protecting against the cellular senescence effects of doxorubicin on healthy cells.

Revealing the Reversal Effect of Galangal (Alpinia galanga L.) Extract Against Oxidative Stress in Metastatic Breast Cancer Cells and Normal Fibroblast Cells Intended as a Co- Chemotherapeutic and Anti-Ageing Agent.

OBJECTIVE: This study intends to explore the potential of galangal extract as a co-chemotherapeutic agent through the analysis of its cytotoxic and migratory effects on metastatic breast cancer cells and as an anti-ageing agent through its senescence inhibitory effect on normal fibroblast cells. METHODS: Galangal ethanolic extract (GE) was subjected to a cytotoxicity test with the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay alone or in combination with doxorubicin (Dox) against 4T1 cells but not in NIH-3T3 cells. Evidence of senescent cells was detected using a SA-ß galactosidase based assay. In addition, the level of reactive oxygen species (ROS), apoptosis, and cell cycle were measured with a flow cytometry-based assay. Meanwhile, cell migration and matrix metalloproteinase (MMP)-9 expression after GE treatment on 4T1 cells were measured using the scratch wound healing assay and gelatin zymography assay, respectively. The metabolomic profiles of GE were traced using gas chromatography-mass spectrometry (GC-MS) analysis. RESULTS: GE effectively inhibited the growth of 4T1 cells with an IC50 value of 135 µg/mL and increased the cytotoxic effect of Dox at concentrations of 50 and 100 µg/mL. GE increased the number of senescent cells arrested in the G2/M phase but did not cause apoptosis. This effect is compounded by increasing intracellular levels of ROS. However, GE reduced senescence to normal in fibroblast cells (NIH 3T3 cells) under oxidative stress by Dox without any changes in the ROS level. Moreover, GE also inhibited the migration of 4T1 cells and suppressed the expression of MMP-9 induced by Dox. CONCLUSION: Galangal has the potential for use as a co-chemotherapeutic agent by inducing senescence in correlation with increasing intracellular ROS toward metastatic breast cancer. However, the effect of GE in decreasing the senescence phenomena toward normal fibroblast cells illustrates its potential as a promising anti-ageing agent.
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