Identification of potential targets of the curcumin analog CCA-1.1 for glioblastoma treatment : integrated computational analysis and in vitro study.

The treatment of glioblastoma multiforme (GBM) is challenging owing to its localization in the brain, the limited capacity of brain cells to repair, resistance to conventional therapy, and its aggressiveness. Curcumin has anticancer activity against aggressive cancers, such as leukemia, and GBM; however, its application is limited by its low solubility and bioavailability. Chemoprevention curcumin analog 1.1 (CCA-1.1), a curcumin analog, has better solubility and stability than those of curcumin. In this study, we explored potential targets of CCA-1.1 in GBM (PTCGs) by an integrated computational analysis and in vitro study. Predicted targets of CCA-1.1 obtained using various databases were subjected to comprehensive downstream analyses, including functional annotation, disease and drug association analyses, protein-protein interaction network analyses, analyses of genetic alterations, expression, and associations with survival and immune cell infiltration. Our integrative bioinformatics analysis revealed four candidate targets of CCA-1.1 in GBM: TP53, EGFR, AKT1, and CASP3. In addition to targeting specific proteins with regulatory effects in GBM, CCA-1.1 has the capacity to modulate the immunological milieu. Cytotoxicity of CCA-1.1 was lower than TMZ with an IC50 value of 9.8 µM compared to TMZ with an IC50 of 40 µM. mRNA sequencing revealed EGFR transcript variant 8 was upregulated, whereas EGFRvIII was downregulated in U87 cells after treatment with CCA-1.1. Furthermore, a molecular docking analysis suggested that CCA-1.1 inhibits EGFR with various mutations in GBM, which was confirmed using molecular dynamics simulation, wherein the binding between CCA-1.1 with the mutant EGFR L861Q was stable. For successful clinical translation, the effects of CCA-1.1 need to be confirmed in laboratory studies and clinical trials.

Comprehensive Computational Analysis of Honokiol Targets for Cell Cycle Inhibition and Immunotherapy in Metastatic Breast Cancer Stem Cells.

Breast cancer stem cells (BCSCs) play a critical role in chemoresistance, metastasis, and poor prognosis of breast cancer. BCSCs are mostly dormant, and therefore, activating them and modulating the cell cycle are important for successful therapy against BCSCs. The tumor microenvironment (TME) promotes BCSC survival and cancer progression, and targeting the TME can aid in successful immunotherapy. Honokiol (HNK), a bioactive polyphenol isolated from the bark and seed pods of Magnolia spp., is known to exert anticancer effects, such as inducing cell cycle arrest, inhibiting metastasis, and overcoming immunotherapy resistance in breast cancer cells. However, the molecular mechanisms of action of HNK in BCSCs, as well as its effects on the cell cycle, remain unclear. This study aimed to explore the potential targets and molecular mechanisms of HNK on metastatic BCSC (mBCSC)-cell cycle arrest and the impact of the TME. Using bioinformatics analyses, we predicted HNK protein targets from several databases and retrieved the genes differentially expressed in mBCSCs from the GEO database. The intersection between the differentially expressed genes (DEGs) and the HNK-targets was determined using a Venn diagram, and the results were analyzed using a protein-protein interaction network, hub gene selection, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, genetic alteration analysis, survival rate, and immune cell infiltration levels. Finally, the interaction between HNK and two HNK-targets regulating the cell cycle was analyzed using molecular docking analysis. The identified potential therapeutic targets of HNK (PTTH) included CCND1, SIRT2, AURKB, VEGFA, HDAC1, CASP9, HSP90AA1, and HSP90AB1, which can potentially inhibit the cell cycle of mBCSCs. Moreover, our results showed that PTTH could modulate the PI3K/Akt/mTOR and HIF1/NFkB/pathways. Overall, these findings highlight the potential of HNK as an immunotherapeutic agent for mBCSCs by modulating the tumor immune environment.

Synthesis and cytotoxicity of the boron carrier pentagamaboronon-0-ol for boron neutron capture therapy against breast cancer.

Boronic acid-containing curcumin analog, pentagamaboronon-0 (PGB-0), acts as a potential boron-carrier agent but has limited water solubility. Thus, a new compound (PGB-0-ol) with better chemical and pharmacological properties than PGB-0 has been synthesized. Molecular docking was performed using a molecular operating environment. Prediction of PGB-0-ol absorption, distribution, metabolism, and excretion (ADME) was performed using pkCSM software. PGB-0-ol was synthesized by adding NaBH4 to PGB-0 and stirring for 1 h. The crude PGB-0-ol was purified using preparative layer chromatography. Cell viability was evaluated using the trypan blue exclusion assay. In comparison to PGB-0 based on molecular docking study, PGB-0-ol could interact in with several cancer biomarkers, such as human epidermal growth factor2 epidermal growth factor receptor, IκB kinase, folate receptor-α, and integrin αvß3. PGB-0-ol also showed an improved ADME profile because of its higher water solubility than PGB-0. PGB-0-ol was synthesized by selective ketone reduction of PGB-0 into primary alcohol by sodium borohydrate producing 30% yield. The cytotoxicity of PGB-0-ol against several breast cancer cells was lower than that of PGB-0. The novel compound PGB-0-ol was synthesized using simple steps. PGB-0-ol has low cytotoxicity against breast cancer cells and could be applied in boron neutron capture therapy as a boron carrier.

Cinnamon oil as a co-chemotherapy agent through inhibition of cell migration and MMP-9 expression on 4T1 cells.

OBJECTIVES: The long-term and high-dose use of doxorubicin as chemotherapy for triple-negative breast cancer (TNBC) patients induces epithelial-to-mesenchymal transition (EMT) and stimulates cancer metastasis. Cinnamaldehyde is a major compound of cinnamon oil (CO) suppressing Snail and NFκB activity that are involved in cell migration. This study aims to explore the activity of CO as a co-chemotherapeutic agent on 4T1 breast cancer cells. METHODS: The CO was obtained by water and steam distillation and was characterized phytochemically by gas chromatography-mass spectrometry (GC-MS). Cytotoxic activity of single CO or in combination with doxorubicin was observed by MTT assay. Cell migration and MMP-9 expression were measured by scratch wound healing and gelatin zymography assays. The intracellular reactive oxygen species (ROS) levels were observed by 2',7'-dichlorofluorescin diacetate (DCFDA) staining flowcytometry. RESULTS: The phytochemical analysis with GC-MS showed that CO contains 14 compounds with cinnamaldehyde as the major compound. CO exhibited cytotoxicity on 4T1 cells with the IC50 value of 25 µg/mL and its combination with doxorubicin decreased cell viability and inhibited cell migration compared to a single use. Furthermore, the combination of CO and doxorubicin inhibited MMP-9 expression and elevated intracellular ROS levels compared to control. CONCLUSIONS: CO has the potential to be developed as a co-chemotherapy agent through inhibition of cell migration, and intracellular ROS levels elevation.

Integrative Bioinformatics Study of Tangeretin Potential Targets for Preventing Metastatic Breast Cancer.

Agents that target metastasis are important to improve treatment efficacy in patients with breast cancer. Tangeretin, a citrus flavonoid, exhibits antimetastatic effects on breast cancer cells, but its molecular mechanism remains unclear. Tangeretin targets were retrieved from PubChem, whereas metastatic breast cancer regulatory genes were downloaded from PubMed. In total, 58 genes were identified as potential therapeutic target genes of tangeretin (PTs). GO and KEGG pathway enrichment analyses of PTs were performed using WebGestalt (WEB-based Gene SeT AnaLysis Toolkit). The PPI network was analyzed using STRING-DB v11.0 and visualized by Cytoscape software. Hub genes were selected on the basis of the highest degree score as calculated by the CytoHubba plugin. Genetic alterations of the PTs were analyzed using cBioPortal. The prognostic values of the PTs were evaluated with the Kaplan-Meier plot. The expression of PTs across breast cancer samples was confirmed using GEPIA. The reliability of the PTs in metastatic breast cancer cells was validated using ONCOMINE. Molecular docking was performed to foresee the binding sites of tangeretin with PIK3Cα, MMP9, PTGS2, COX-2, and IKK. GO analysis showed that PTs participate in the biological process of stimulus response, are the cellular components of the nucleus and the membrane, and play molecular roles in enzyme regulation. KEGG pathway enrichment analysis revealed that PTs regulate the PI3K/Akt pathway. Genetic alterations for each target gene were MTOR (3%), NOTCH1 (4%), TP53 (42%), MMP9 (4%), NFKB1 (3%), PIK3CA (32%), PTGS2 (15%), and RELA (5%). The Kaplan-Meier plot showed that patients with low mRNA expression levels of MTOR, TP53, MMP9, NFKB1, PTGS2, and RELA and high expression of PIK3CA had a significantly better prognosis than their counterparts. Further validation of gene expression by using GEPIA revealed that the mRNA expression of MMP9 was significantly higher in breast cancer tissues than in normal tissues, whereas the mRNA expression of PTGS2 showed the opposite. Analysis with ONCOMINE demonstrated that the mRNA expression levels of MMP9 and NFKB1 were significantly higher in metastatic breast cancer cells than in normal tissues. The results of molecular docking analyses revealed the advantage of tangeretin as an inhibitor of PIK3CA, MMP9, PTGS2, and IKK. Tangeretin inhibits metastasis in breast cancer cells by targeting TP53, PTGS2, MMP9, and PIK3CA and regulating the PI3K/Akt signaling pathway. Further investigation is needed to validate the results of this study.

Identification of potential therapeutic target of naringenin in breast cancer stem cells inhibition by bioinformatics and in vitro studies.

Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells' sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein-protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.

Cellular uptake evaluation of pentagamaboronon-0 (PGB-0) for boron neutron capture therapy (BNCT) against breast cancer cells.

Pentagamaboronon-0 (PGB-0), a curcumin analog compound, has been synthesized as a candidate of boron-carrier pharmaceutical (BCP) for boron neutron capture therapy (BNCT); however, this compound is poorly soluble in water. To improve its solubility, aqueous formulations of PGB-0 with a monosaccharide, fructose or sorbitol, were successfully synthesized, namely PGB-0-F and PGB-0-So, respectively. The cytotoxicity study showed that PGB-0-F and PGB-0-So exerted low cytotoxicity against MCF-7 and MDA-MB 231 breast cancer cells. The cellular uptake study using inductively coupled plasma optical emission spectrometry (ICP-OES) and DAHMI live-cell imaging indicated that these compounds were accumulated and distributed within the cytoplasm and cell nuclei. The cellular uptake mechanism was also evaluated to clarify the contribution of the glucose transporter, and the results demonstrated that these compounds entered through active transport into MCF-7 cells but through passive diffusion into MDA-MB 231 cells. In conclusion, the sugar formulations of PGB-0 only improved PGB-0 solubility but had no role in its cellular uptake.

Cardioprotective and hepatoprotective effects of Citrus hystrix peels extract on rats model.

OBJECTIVE: To observe the combination effect of doxorubicin and Citrus hystrix (kaffir lime's) peel ethanolic extract (ChEE) on blood serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity and cardio-hepato-histopathology of female Sprague Dawley rats. METHODS: Doxorubicin and ChEE (5 rats per group) were administered in five groups of 3 rats each for 11 d. Group I: doxorubicin (dox) 4.67 mg/kg body weight; Group II: dox+ChEE 500 mg/kg body weight; Group III: dox+ChEE 1 000 mg/kg body weight; Group IV: ChEE 1 000 mg/kg body weight; Group V: untreated (control). RESULTS: ChEE repaired cardiohistopathology profile of doxorubicin induced cardiotoxicity and hepatotoxicity rats, but did not repair neither hepatohistopathology profile nor reduce serum activity of ALT and AST. CONCLUSION: ChEE has potency to be developed as cardioprotector agent in chemotherapy.

Synergistic effects of ethyl acetate fraction of Ficus septica Burm. f. and doxorubicin chemotherapy on T47D human breast cancer cell line.

OBJECTIVE: Previously, ethanolic extract of Ficus septica Burm. f. (Moraceae) leaves and its ethyl acetate soluble fraction (EASF) exhibited potent cytotoxic effects on T47D breast cancer cells. In the present study, we further investigated the effects of EASF of ethanolic extract of F. septica leaves in combination with doxorubicin on T47D breast cancer cell line in cytotoxicity, cell cycle arrest and apoptosis induction. METHODS: The cytotoxic effect analysis on T47D cells was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Analysis of cell cycle distribution was performed using a flowcytometer and the data were analyzed using ModFit LT 3.0 program. Apoptosis assay was carried out by double staining method using ethidium bromide-acridin orange. The expression of cleaved-poly ADP-ribose polymerase in the T47D cell line was identified using immunohistochemical techniques. RESULTS: The combination of doxorubicin (2 to 8 nmol/L) with EASF (0.875 to 7 µg/mL) more potently inhibited cell growth than the single treatment of doxorubicin in T47D cells. In addition, the combination of doxorubicin and EASF could increase the incidence of cells undergoing apoptosis. EASF was found to improve cytotoxic effect of doxorubicin by changing the inhibition of cell cycle G(2)/M to G(1) phase. The combination also exhibited a more intensive stimulatory effect on cleaved-PARP expression in T47D cells than the single treatment. CONCLUSION: It is concluded that EASF may enhance doxorubicin activities in T47D cells by inducing apoptosis and cell cycle arrest.

Natural products for cancer-targeted therapy: citrus flavonoids as potent chemopreventive agents.

Targeted therapy has been a very promising strategy of drug development research. Many molecular mechanims of diseases have been known to be regulated by abundance of proteins, such as receptors and hormones. Chemoprevention for treatment and prevention of diseases are continuously developed. Pre-clinical and clinical studies in chemoprevention field yielded many valuable data in preventing the onset of disease and suppressing the progress of their growth, making chemoprevention a challenging and a very rational strategy in future researches. Natural products being rich of flavonoids are those fruits belong to the genus citrus. Ethanolic extract of Citrus reticulata and Citrus aurantiifolia peels showed anticarcinogenic, antiproliferative, co-chemotherapeutic and estrogenic effects. Several examples of citrus flavonoids that are potential as chemotherapeutic agents are tangeretin, nobiletin, hesperetin, hesperidin, naringenin, and naringin. Those flavonoids have been shown to possess inhibition activity on certain cancer cells' growth through various mechanisms. Moreover, citrus flavonoids also perform promising effect in combination with several chemotherapeutic agents against the growth of cancer cells. Some mechanisms involved in those activities are through cell cycle modulation, antiangiogenic effect, and apoptosis induction. Previous studies showed that tangeretin suppressed the growth of T47D breast cancer cells by inhibiting ERK phosphorylation. While in combination with tamoxifen, doxorubicin, and 5-FU, respectively, it was proven to be synergist on several cancer cells. Hesperidin and naringenin increased cytotoxicitity of doxorubicin on MCF-7 cells and HeLa cells. Besides, citrus flavonoids also performed estrogenic effect in vivo. One example is hesperidin having the ability to decrease the concentration of serum and hepatic lipid and reduce osteoporosis of ovariectomized rats. Those studies showed the great potential of citrus fruits as natural product to be developed as not only the source of co-chemotherapeutic agents, but also phyto-estrogens. Therefore, further study needs to be conducted to explore the potential of citrus fruits in overcoming cancer.

Immunomodulatory effects of hexane insoluble fraction of Ficus septica Burm. F. in doxorubicin-treated rats.

The use of chemotherapeutics induces cardiotoxicity and affects immune functions, therefore development of combinatorial agents against cardiotoxicity and immunosuppression needs to be explored. Previous studies of the hexane insoluble fraction (HIF) of an ethanolic extract of Ficus septica leaves showed anticancer effects singly and in combination with doxorubicin on T47D breast cancer cells. In this present study, it was evaluated for its immunomodulatory activities in doxorubicin-treated rats. Thirty male Sprague Dawley rats were divided into five groups consisting of six rats each as follows: Group 1, receiving oral saline 10 ml/kg BW (control group); Group 2, receiving HIF dose 750 mg/kg BW orally, once daily; Group 3, receiving HIF dose 1.500 mg/kg BW orally, once daily; Group 4, given oral saline 10 ml/kg BW (normal group); Group 5, receiving HIF dose 1.500 mg/kg BW orally, once daily. The rats of group 1-3 were intramuscularly administered with doxorubicin at a dose of 4.67 mg/kg BW at the days 1 and 4 to suppress immune functions. Concomitantly, the rats were treated with saline or HIF for seven consecutive days (1 to 7). Treatment of HIF succeeded in reducing side effects of doxorubicin based on increasing lymphocyte density and phagocytosis activity and capacity of macrophages, as well as increasing the CD8+blood level and decreasing spleen IL-10 expression. Hexane insoluble fraction of of ethanolic extract of Ficus septica leaves has potential as a protective agent combined with doxorubicin.