The Phytochemical Scoulerine Inhibits Aurora Kinase Activity to Induce Mitotic and Cytokinetic Defects.

To identify novel bioactive compounds, an image-based, cell culture screening of natural product extracts was conducted. Specifically, our screen was designed to identify phytochemicals that might phenocopy inhibition of the chromosomal passenger protein complex in eliciting mitotic and cytokinetic defects. A known alkaloid, scoulerine, was identified from the rhizomes of the plant Corydalis decumbens as being able to elicit a transient mitotic arrest followed by either apoptosis induction or polyploidy. In examining the mitotic abnormality further, we observed that scoulerine could elicit supernumerary centrosomes during mitosis, but not earlier in the cell cycle. The localization of NUMA1 at spindle poles was also inhibited, suggesting diminished potential for microtubule recruitment and spindle-pole focusing. Polyploid cells emerged subsequent to cytokinetic failure. The concentration required for scoulerine to elicit all its cell division phenotypes was similar, and an examination of related compounds highlighted the requirement for proper positioning of a hydroxyl and a methoxy group about an aromatic ring for activity. Mechanistically, scoulerine inhibited AURKB activity at concentrations that elicited supernumerary centrosomes and polyploidy. AURKA was only inhibited at higher concentrations, so AURKB inhibition is the likely mechanism by which scoulerine elicited division defects. AURKB inhibition was never complete, so scoulerine may be a suboptimal AURK inhibitor or work upstream of the chromosomal passenger protein complex to reduce AURKB activity. Scoulerine inhibited the viability of a variety of human cancer cell lines. Collectively, these findings uncover a previously unknown activity of scoulerine that could facilitate targeting human cancers. Scoulerine, or a next-generation analogue, may be useful as a nontoxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.

Enhancing radiosensitisation of BRCA2-proficient and BRCA2-deficient cell lines with hyperthermia and PARP1-i.

Poly(ADP-ribose)polymerase1 (PARP1) is an important enzyme in regulating DNA replication. Inhibition of PARP1 can lead to collapsed DNA forks which subsequently causes genomic instability, making DNA more susceptible in developing fatal DNA double strand breaks. PARP1-induced DNA damage is generally repaired by homologous recombination (HR), in which BRCA2 proteins are essential. Therefore, BRCA2-deficient tumour cells are susceptible to treatment with PARP1-inhibitors (PARP1-i). Recently, BRCA2 was shown to be down-regulated by hyperthermia (HT) temporarily, and this consequently inactivated HR for several hours. In this study, we investigated whether HT exclusively interferes with HR by analysing thermal radiosensitisation of BRCA2-proficient and deficient cells. After elucidating the equitoxicity of PARP1-i on BRCA2-proficient and deficient cells, we studied the cell survival, apoptosis, DNA damage (γ-H2AX foci and comet assay) and cell cycle distribution after different treatments. PARP1-i sensitivity strongly depends on the BRCA2 status. BRCA2-proficient and deficient cells are radiosensitised by HT, indicating that HT does not exclusively act by inhibition of HR. In all cell lines, the addition of HT to radiotherapy and PARP1-i resulted in the lowest cell survival, the highest levels of DNA damage and apoptotic levels compared to duo-modality treatments. Concluding, HT not only inhibits HR, but also has the capability of radiosensitising BRCA2-deficient cells. Thus, in case of BRCA2-mutation carriers, combining HT with PARP1-i may boost the treatment efficacy. This combination therapy would be effective for all patients with PARP1-i regardless of their BRCA status.

Ellagic Acid Enhances Apoptotic Sensitivity of Breast Cancer Cells to γ-Radiation.

Herbal polyphenols have gained increased significance because of the promises they hold in the prevention and treatment of cancer. There exists an enormous opportunity for the screening and valuation of natural dietary compounds in the development of an effective chemopreventive drug and radiosensitizer that may be of practical use for patients undergoing cancer therapy. This study describes the effect of the flavonoid ellagic acid (EA) on gamma-irradiated human breast cancer MCF-7 cells in vitro when administered alone or in combination with radiation. It was interesting to find the radioprotective effect of EA on NIH3T3, which is a normal cell line. Irradiation of breast tumor cells in the presence of EA (10 µM) to doses of 2 and 4-Gy gamma radiation produced a marked synergistic tumor cytotoxicity while it was found to aid recovery from the radiation damage to NIH3T3 cells. When cells were given a combined treatment of EA and radiation, the cell death increased to 21.7% and 20.7% in the 2 and 4-Gy-treated cells respectively, significantly (P < 0.05) reducing the capacity of MCF-7 cells to form colonies. Even at 24 h, 38 foci/cell were observed in samples that were given the combined treatment, suggesting the cells' inability in repairing the damage. Also, increased apoptosis in EA+ 2Gy (50%) and EA+ 4 Gy (62%)-treated cells was observed in the the sub-G1 phase of the cell cycle. A 6.2-fold decrease in the mitochondrial membrane potential was observed in the combined treatment of EA and IR that facilitated the upregulation of pro-apopttotic Bax and downregulation of Bcl-2, pushing the MCF-7 cells to undergo an apoptotic cell death. It is suggested that EA may be a potential drug adjuvant for improving cancer radiotherapy by increasing tumor toxicity and reducing the normal cell damage caused by irradiation.

The phytochemical, corynoline, diminishes Aurora kinase B activity to induce mitotic defect and polyploidy.

Plants are a rich source for bioactive compounds. However, plant extracts can harbor a mixture of bioactive molecules that promote divergent phenotypes and potentially have confounding effects in bioassays. Even with further purification and identification, target deconvolution can be challenging. Corynoline and acetylcorynoline, are phytochemicals that were previously isolated through a screen for compounds able to induce mitotic arrest and polyploidy in oncogene expressing retinal pigment epithelial (RPE) cells. Here, we shed light on the mechanism by which these phytochemicals can attack human cancer cells. Mitotic arrest was coincident to the induction of centrosome amplification and declustering, causing multi-polar spindle formation. Corynoline was demonstrated to have true centrosome declustering activity in a model where A549 cells were chemically induced to have more than a regular complement of centrosomes. Corynoline could inhibit the centrosome clustering required for pseudo-bipolar spindle formation in these cells. The activity of AURKB, but not AURKA or polo-like kinase 4, was diminished by corynoline. It only partially inhibited AURKB, so it may be a partial antagonist or corynoline may work upstream on an unknown regulator of AURKB activity or localization. Nonetheless, corynoline and acetylcorynoline inhibited the viability of a variety of human cancer derived cell lines. These phytochemicals could serve as prototypes for a next-generation analog with improved potency, selectivity or in vivo bioavailability. Such an analog could be useful as a non-toxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.

Augmentation of radiation-induced apoptosis by ellagic acid.

This study evaluates the potential of ellagic acid (EA) as an enhancer of radiation-induced apoptosis in cancer cells. HeLa cells treated with EA and gamma radiation showed increased superoxide generation, upregulated p53 protein expression, and decreased antioxidant enzymes. We also found that EA and radiation enhance capase-3 activity via oxidative stress, increased intracellular calcium levels, and phospholipase C and cause a drop in mitochondrial potential. These results might provide a basis for prominent reduction of cancer cell using EA as an adjunct to radiotherapy and an opportunity to lower the toxic radiation doses to improve the quality of life.

A multimodal treatment of carbon ions irradiation, miRNA-34 and mTOR inhibitor specifically control high-grade chondrosarcoma cancer stem cells.

BACKGROUND AND PURPOSE: High-grade chondrosarcomas are chemo- and radio-resistant cartilage-forming tumors of bone that often relapse and metastase. Thus, new therapeutic strategies are urgently needed. MATERIAL AND METHODS: Chondrosarcoma cells (CH-2879) were exposed to carbon-ion irradiation, combined with miR-34 mimic and/or rapamycin administration. The effects of treatment on cancer stem cells, stemness-associated phenotype, radioresistance and tumor-initiating properties were evaluated. RESULTS: We show that high-grade chondrosarcoma cells contain a population of radioresistant cancer stem cells that can be targeted by a combination of carbon-ion therapy, miR-34 mimic administration and/or rapamycin treatment that triggers FOXO3 and miR-34 over-expression. mTOR inhibition by rapamycin triggered FOXO3 and miR-34, leading to KLF4 repression. CONCLUSION: Our results show that particle therapy combined with molecular treatments effectively controls cancer stem cells and may overcome treatment resistance of high-grade chondrosarcoma.

Studying the in Silico Effect of Ellagic Acid on HIF-2α to Improve Efficacy of Anticancer Therapy.

The hypoxic tumor microenvironment is one of the major causes of the enhanced chemoresistant and radioresistant behavior of cancer cells. Therefore, the hypoxia-induced factor (HIF) pathway can be endorsed, for not only the malignant phenotype of the cells, but also its metastatic potential. Many drugs targeting the HIF pathways have failed in the clinical setting to demonstrate therapeutic efficacy. Such failures occur due to lack of specificity or redundancy in the complexity of tumor signaling/metabolism that can overcome the inhibitory effects. Another important factor is the letdown of the compound that can be accredited to lack of patient selection in the trials. Although many clinical trials have evaluated the efficacy of anticancer therapeutics and examined their effects on HIF levels, patients were not selected based on their HIF expression levels. If patients do not have elevated levels of HIF, then the therapeutics that target the HIF pathway may be less effective. In the present work, we have targeted HIF-2α of the HIF pathway. Ellagic acid (EA), a well-known anticancer compound and radiosensitizer, is used to inhibit the activity of HIF-2α. Our results show a very unique binding of EA with HIF-2α. Such new agents should be used in combination therapy and will hopefully overcome the resistance that may develop during initial treatment if the patient is identified to have enhanced expression of HIF-2α. Molecular dynamics studies followed solvation free energy calculations (molecular mechanics Poisson-Boltzmann surface area) for understanding the binding stability and per residue contribution. Our in silico data look promising and EA should be studied more in in vitro and in vivo for further analysis of its efficacy.

Enhancing synthetic lethality of PARP-inhibitor and cisplatin in BRCA-proficient tumour cells with hyperthermia.

BACKGROUND: Poly-(ADP-ribose)-polymerase1 (PARP1) is involved in repair of DNA single strand breaks. PARP1-inhibitors (PARP1-i) cause an accumulation of DNA double strand breaks, which are generally repaired by homologous recombination (HR). Therefore, cancer cells harboring HR deficiencies are exceptionally sensitive to PARP1-i. For patients with HR-proficient tumors, HR can be temporarily inhibited by hyperthermia, thereby inducing synthetic lethal conditions in every tumor type. Since cisplatin is successfully used combined with hyperthermia (thermochemotherapy), we investigated the effectiveness of combining PARP1-i with thermochemotherapy. RESULTS: The in vitro data demonstrate a decreased in cell survival after addition of PARP1-i to thermochemotherapy, which can be explained by increased DNA damage induction and less DSB repair. These in vitro findings are in line with in vivo model, in which a decreased tumor growth is observed upon addition of PARP1-i. MATERIALS AND METHODS: Survival of three HR-proficient cell lines after cisplatin, hyperthermia and/or PARP1-i was studied. Cell cycle analyses, quantification of γ-H2AX foci and apoptotic assays were performed to understand these survival data. The effects of treatments were further evaluated by monitoring tumor responses in an in vivo rat model. CONCLUSIONS: Our results in HR-proficient cell lines suggest that PARP1-i combined with thermochemotherapy can be a promising clinical approach for all tumors independent of HR status.

Inhibition of the p53 Y220C Mutant by 1-Hydroxy-2- Methylanthraquinone Derivatives: A Novel Strategy for Cancer Therapy.

Y220C, a substitution mutation in p53, causes major structural changes in the protein and is known to form a new protein cavity. This cavity is reckoned to accommodate small drug candidates that may play a key role in cancer treatment. Present study was aimed at determining a drug candidate that could inhibit the mutant p53 based on structural drug rationale. Docking of mutated p53 was performed to determine the drug of choice from the derivatives of 1-hydroxy-2- methylanthraquinone exhibiting anti-cancer properties. The cavity had been tested for identification of an accurate position vector for molecular docking studies using structure based drug design. The docked structure was validated using discovery studio 3.5. The best choice of two molecules were obtained by docking in specific solvent for 6 nanoseconds at a temperature of 310 K. Out of a library of compounds, acetamido-2-carboxy-4-dimethylamino-2- hydroxybenzophenone satisfied the ADMET and was found to be a potential target for mutant p53. This ligand binds at the active site of the protein. Results of present study offer a rationale of the lead ligands that can rescue oncogenic p53 by targeting the mutation site. Therefore, it is suggestive that small molecules may serve as an effective and novel anti-cancer drug.

On the mechanism of cellular toxicity in breast cancer by ionizing radiation and chemotherapeutic drugs.

Breast cancer is the second leading cause of cancer mortality and the most frequent cancer found in women around the globe. The development of breast cancer is a multistep and complicated process that includes the development of ductal and lobular cells into atypical hyperplasia, carcinoma in situ, and invasive carcinoma, with an ability to metastasize. The efficacy of radiotherapy in breast cancer seems to be reduced because of a frequently observed lack of cellular sensitivity to apoptosis. Both Bcl-2 and p53 are linked to apoptosis pathways and are known to play a role in the outcome of radiotherapy. Resistance of tumor cells to therapeutic drugs and the undesirable cytotoxicity of normal cells are frequently observed in treatment outcomes in clinics. Research is, therefore, needed to develop strategies for improving the protocols of chemotherapy and radiotherapy in patients with breast cancer. This review focuses on understanding the molecular mechanisms of enhanced tumor cell killing by the combined action of certain anticancer drugs together with gamma radiation in vitro, with possible implications for practical applications in clinics.