Identification of Gedunin from a Phytochemical Depository as a Novel Multidrug Resistance-Bypassing Tubulin Inhibitor of Cancer Cells.

The chemotherapy of tumors is frequently limited by the development of resistance and severe side effects. Phytochemicals may offer promising candidates to meet the urgent requirement for new anticancer drugs. We screened 69 phytochemicals, and focused on gedunin to analyze its molecular modes of action. Pearson test-base correlation analyses of the log10IC50 values of 55 tumor cell lines of the National Cancer Institute (NCI), USA, for gedunin with those of 91 standard anticancer agents revealed statistically significant relationships to all 10 tested microtubule inhibitors. Thus, we hypothesized that gedunin may be a novel microtubule inhibitor. Confocal microscopy, cell cycle measurements, and molecular docking in silico substantiated our assumption. Agglomerative cluster analyses and the heat map generation of proteomic data revealed a subset of 40 out of 3171 proteins, the expression of which significantly correlated with sensitivity or resistance for the NCI cell line panel to gedunin. This indicates the complexity of gedunin's activity against cancer cells, underscoring the value of network pharmacological techniques for the investigation of the molecular modes of drug action. Finally, we correlated the transcriptome-wide mRNA expression of known drug resistance mechanism (ABC transporter, oncogenes, tumor suppressors) log10IC50 values for gedunin. We did not find significant correlations, indicating that gedunin's anticancer activity might not be hampered by classical drug resistance mechanisms. In conclusion, gedunin is a novel microtubule-inhibiting drug candidate which is not involved in multidrug resistance mechanisms such as other clinically established mitotic spindle poisons.

Biomarker Profiling Revealed Carcinoembryonic Antigen as a Target of Artesunate in a Ductal Breast Cancer Patient.

BACKGROUND/AIM: Patients with metastatic tumors commonly have a poor prognosis. Frequently, patients suffering from progressive tumors have a high willingness for the compassionate use of non-approved medications. One of these medications is the antimalarial drug artesunate (ART) which also showed profound anticancer activity in vitro, in vivo, and in preliminary clinical pilot studies. Herein, we report on the compassionate use of ART in a patient with metastatic breast cancer. PATIENTS AND METHODS: The clinical course of a Caucasian female who was diagnosed with ductal breast cancer at the age of 33 is described. Tumor markers in the blood have been measured, and tumor-associated protein expression has been determined by immunohistochemistry. Microscale thermophoresis and molecular docking in silico were used to study protein-drug interactions. RESULTS: The tumor responded to ART administered at doses of 150-300 mg daily, and the patient experienced a stabilization of her disease for 1.5 years. ART treatment caused no or minimal side-effects (headache, dizziness, slight tachycardia, slight stomach upset, slight fatigue). Tumor marker determination in the blood of the patient revealed a reduction of carcinoembryonic antigen (CEA), but not CA 27.29 or CA 15.3 levels. We hypothesized that the reduction of CEA levels might be due to binding of ART to this protein. Microscale thermophoresis with recombinant CEA indeed showed binding of ART to this protein in vitro. This result was verified by molecular docking in silico. Immunohistochemical biomarker profiling and computerbased quantification of biomarker expression in a tumor biopsy revealed strong expression of COX2, GRP78, CD71, GSTP1, and c-MYC but weak or minimal expression of VEGFR, P-glycoprotein, survivin, and LOX1. CONCLUSION: Among a panel of tumor-related proteins tested, the interaction with CEA may have contributed to the anticancer activity of ART in this patient. It deserves further investigation whether CEA represents not only a valuable biomarker but also a treatment target. ART might be useful for the individualized treatment of metastatic breast tumors.

Identification of active components in Andrographis paniculata targeting on CD81 in esophageal cancer in vitro and in vivo.

BACKGROUND: Esophageal cancer (EC) is highly prevalent in Eastern Asia (including China) with high rates of mortality. The metastatic tendency in EC is associated with a poor prognosis. Our previous studies have demonstrated the suppressive effects of Andrographis paniculata water extract (APW) on metastatic esophageal cancer in vitro and in tumor-bearing mice models, as well as illustrated the potential underlying mechanism by transcriptome analysis. HYPOTHESIS: High expressions of several membrane protein tetraspanins were reported to lead to a high risk of metastasis in esophageal cancer in patients. We hypothesized that APW could downregulate the expression of tetraspanin CD81 in esophageal cancer cells and xenografts. METHODS: Human esophageal cancer cells EC109 and KYSE520 were incubated with APW for 24 hours in cell culture, while mice bearing EC109 xenograft tumors were treated with APW for 21 days. The expressions of CD81 in cancer cells and in tumors from mice were evaluated. Molecular docking and microscale thermophoresis analyses were applied to identify the components in APW interacting with CD81. The influence of the identified components on CD81 expression was further evaluated in EC109 cells. RESULTS: APW could significantly suppress the expressions of CD81 in both EC109 and KYSE520 cells in a concentration-dependent manner. Treatment of APW in xenograft-bearing mice reduces the metastasis in lungs, livers, and lymph nodes. The expression of CD81 in xenograft tumors of APW-treated mice was significantly lower than those of untreated control mice. The binding of andrographolide, bisandrographolide A, and bisandrographolide C with CD81 were elucidated by microscale thermophoresis. The suppressive effects of these compounds on the motility of EC109 cells, as well as CD81 protein and mRNA expressions, were further confirmed. CONCLUSION: This is the first time to demonstrate that andrographolide, bisandrographolide A, and bisandrographolide C, which are present in APW, bind to CD81 and suppress its function. These compounds are likely to be responsible for the anti-metastatic activities of APW in esophageal cancer.

In Silico and In Vitro Screening of 50 Curcumin Compounds as EGFR and NF-κB Inhibitors.

The improvement of cancer chemotherapy remains a major challenge, and thus new drugs are urgently required to develop new treatment regimes. Curcumin, a polyphenolic antioxidant derived from the rhizome of turmeric (Curcuma longa L.), has undergone extensive preclinical investigations and, thereby, displayed remarkable efficacy in vitro and in vivo against cancer and other disorders. However, pharmacological limitations of curcumin stimulated the synthesis of numerous novel curcumin analogs, which need to be evaluated for their therapeutic potential. In the present study, we calculated the binding affinities of 50 curcumin derivatives to known cancer-related target proteins of curcumin, i.e., epidermal growth factor receptor (EGFR) and nuclear factor κB (NF-κB) by using a molecular docking approach. The binding energies for EGFR were in a range of −12.12 (±0.21) to −7.34 (±0.07) kcal/mol and those for NF-κB ranged from −12.97 (±0.47) to −6.24 (±0.06) kcal/mol, indicating similar binding affinities of the curcumin compounds for both target proteins. The predicted receptor-ligand binding constants for EGFR and curcumin derivatives were in a range of 0.00013 (±0.00006) to 3.45 (±0.10) µM and for NF-κB in a range of 0.0004 (±0.0003) to 10.05 (±4.03) µM, indicating that the receptor-ligand binding was more stable for EGFR than for NF-κB. Twenty out of 50 curcumin compounds showed binding energies to NF-κB smaller than −10 kcal/mol, while curcumin as a lead compound revealed free binding energies of >−10 kcal/mol. Comparable data were obtained for EGFR: 15 out of 50 curcumin compounds were bound to EGFR with free binding energies of <−10 kcal/mol, while the binding affinity of curcumin itself was >−10 kcal/mol. This indicates that the derivatization of curcumin may indeed be a promising strategy to improve targe specificity and to obtain more effective anticancer drug candidates. The in silico results have been exemplarily validated using microscale thermophoresis. The bioactivity has been further investigated by using resazurin cell viability assay, lactate dehydrogenase assay, flow cytometric measurement of reactive oxygen species, and annexin V/propidium iodide assay. In conclusion, molecular docking represents a valuable approach to facilitate and speed up the identification of novel targeted curcumin-based drugs to treat cancer.