Cancer is one of the leading causes of death worldwide, with a mortality rate of more than 9 million deaths reported in 2018. Conventional anti-cancer therapy can greatly improve survival however treatment resistance is still a major problem especially in metastatic disease. Targeted anti-cancer therapy is increasingly used with conventional therapy to improve patients’ outcomes in advanced and metastatic tumors. However, due to the complexity of cancer biology and metastasis, it is urgent to develop new agents and evaluate the anti-cancer efficacy of available treatments. Many phytochemicals from medicinal plants have been reported to possess anti-cancer properties. One such compound is known as oridonin, a bioactive component of Rabdosia rubescens. Several studies have demonstrated that oridonin inhibits angiogenesis in various types of cancer, including breast, pancreatic, lung, colon and skin cancer. Oridonin’s anti-cancer effects are mediated through the modulation of several signaling pathways which include upregulation of oncogenes and pro-angiogenic growth factors. Furthermore, oridonin also inhibits cell migration, invasion and metastasis via suppressing epithelial-to-mesenchymal transition and blocking downstream signaling targets in the cancer metastasis process. This review summarizes the recent applications of oridonin as an anti-angiogenic and anti-metastatic drug both in vitro and in vivo, and its potential mechanisms of action.

Summary: Radiotherapy is an effective treatment modality for breast cancer but, unfortunately, not all patients respond fully with a significant number experiencing local recurrences. Overexpression of thioredoxin and thioredoxin reductase has been reported to cause multidrug and radiation resistance - their inhibition may therefore improve therapeutic efficacy. Novel indolequinone compounds have been shown, in pancreatic cancer models, to inhibit thioredoxin reductase activity and exhibit potent anticancer activity. The present study evaluates, using in vitro breast cancer models, the efficacy of a novel indolequinone compound (IQ9) as a single agent and in combination with ionising radiation using a variety of endpoint assays including cell proliferation, clonogenic survival, enzyme activity, and western blotting. Three triple-negative breast cancer (MDA-MB-231, MDA-MB-468, and MDA-MB-436) and two luminal (MCF-7 and T47D) breast cancer cell lines were used. Results show that treatment with IQ9 significantly inhibited thioredoxin reductase activity, and inhibited cell growth and colony formation of breast cancer cells with IC₅₀ values in the low micromolar ranges. Enhanced radiosensitivity of triple-negative breast cancer cells was observed, with sensitiser enhancement ratios of 1.20–1.43, but with no evident radiosensitisation of luminal breast cancer cell lines. IQ9 upregulated protein expression of thioredoxin reductase in luminal but not in triple-negative breast cancer cells which may explain the observed differential radiosensitisation. This study provides important evidence of the roles of the thioredoxin system as an exploitable radiobiological target in breast cancer cells and highlights the potential therapeutic value of indolequinones as radiosensitisers.***This study was not part of a clinical trial. Clinical trial registration number: N/A

Hypoxia plays a significant role in solid tumors by the increased expression of hypoxia-inducible factor-1α (HIF-1α), which is known to promote cancer invasion and metastasis. Cancer-cell invasion dynamically begins with the degradation of the extracellular matrix (ECM) via invadopodia formation. The chemical substrates that are utilized by hypoxic cells as fuel to drive invadopodia formation are still not fully understood. Therefore, the aim of the study was to maintain MDA-MB-231 cells under hypoxia conditions to allow cells to form a large number of invadopodia as a model, followed by identifying their nutrient utilization. The results of the study revealed an increase in the number of cells forming invadopodia under hypoxia conditions. Moreover, Western blot analysis confirmed that essential proteins for hypoxia and invadopodia, including HIF-1α, vascular endothelial growth factor (VEGF), metallopeptidase-2 (MMP-2), and Rho guanine nucleotide exchange factor 7 (β-PIX), significantly increased under hypoxia. Interestingly, phenotype microarray showed that only 11 chemical substrates from 367 types of substrates were significantly metabolized in hypoxia compared to in normoxia. This is thought to be fuel for hypoxia to drive the invasion process. In conclusion, we found 11 chemical substrates that could have potential energy sources for hypoxia-induced invadopodia formation of these cells. This may in part be a target in the hypoxic tumor and invadopodia formation. Additionally, these findings can be used as potential carrier targets in cancer-drug discovery, such as the usage of dextrin.

Background: Breast cancer is the most common cancer in women in the United Kingdom. The glutathione (GSH) system is a major redox buffering system involved in regulating radioresponse of cancer cells. Overexpression has been shown to cause multidrug and radiation resistance. Its modulation may increase radiosensitivity and improve radiotherapy efficacy. Piperlongumine (PL) can reportedly inhibit GSH system function and reduce proliferation of a variety cancer cells in vitro. The current study investigates novel PL analogues to determine efficacy in triple-negative (TNBC) and luminal breast cancer cell lines, as both single agents and in combination with ionising radiation. Methods: Two novel PL analogues (LH91 and LH92) were used along with the parental drug, PL. Cell proliferation assays were conducted in MDA-MB-231 (TNBC) and T47D (luminal) lines at various drug concentrations and times. At 48hr post treatment, cells were also assessed for clonogenic survival. Clonogenic radiosensitisation was assessed by treating with PL agents or L-buthionine-S, R-sulfoximine (BSO, a well characterised GSH radiosensitiser) for 48hr then irradiation with 0-8Gy X-rays. ROS levels were evaluated using H₂DCFDA flow cytometry following IC₅₀ drug treatment alone or with subsequent exposure to 1mM H₂O₂ for 1hr. Results: PL, LH91 and LH92 inhibited proliferation and decreased clonogenic survival in both lines. From proliferation assays, MDA-MB-231 cells were more sensitive to PL at 48hr (P=0.047) and 72hr (P=0.01) with an IC₅₀ dose of approx. 4μM versus approx. 12μM in T47D’s. There was a significant difference between IC₅₀ doses of LH91 in MDA-MB-231 (5μM) and T47D (12μM) cell lines at 72hr (P=0.006), however this was not observed at 48hr treatment (P=0.178). In contrast, there was no differential sensitivity of LH92 in cell proliferation between the two cell lines at 48 and 72hr. Interestingly, in clonogenic assays, MDA-MB-231’s were more sensitive to LH92 (P=0.01) with an IC₅₀ of 4μM versus 11μM in T47D’s. There were no significant differences in clonogenic survival between each line after treatment with PL or LH91. No radiosensitisation was observed with PL or PL analogues in MDA-MBA-231 however BSO, as a drug comparator, enhanced radiation response with a sensitizer enhancement ratio (SER) of 1.13 at 1% iso-survival. Radiation and drug combinations in T47D cells are ongoing. There were no changes in ROS levels with the drug alone in MDA-MB-231; however, with subsequent exposure of H₂O₂, there was a 1.7-fold increase in ROS level with LH91 at 48hr (P=0.002). Conclusion: The TNBC cell line is more sensitive to the cytotoxic effect of PL agents (PL and LH91 in cell proliferation and LH92 in clonogenic assays) in comparison to the luminal cell line; however no altered radiosensitisation was observed. The mechanism of such differential sensitivity is yet to be determined. Note: This abstract was not presented at the meeting. Citation Format: Nurul Akmaryanti Abdullah, John Moses, Li Chen Han, Sarah Storr, Aula Ammar, Stewart G. Martin. Differential cytotoxic effects of Piperlongumine analogues and their radiotherapeutic response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5210. doi:10.1158/1538-7445.AM2017-5210