Hypoxia, a condition characterised by low oxygen levels, leads to increased production of a protein called hypoxia-inducible factor-1 alpha (HIF-1α) in cancer cells. This protein is involved in driving processes such as vascularization, cytoskeletal reorganisation, and epithelial-to-mesenchymal transformation (EMT), which contribute to metastasis. Previous studies used hypoxic workstations, chambers, and incubators to evaluate the effects of hypoxia on colon cancer cell lines. In a cell culture model, hypoxic conditions can also be induced using dimethyloxalylglycine (DMOG) as the hypoxia-mimicking agent. This study aims to investigate the effects of DMOG-induced hypoxia on colon cancer metastasis, focusing on cell migration and invasion. HCT116 cells were subjected to hypoxic conditions by treating them with DMOG, and the expression of HIF-1α proteins was measured at various time points, followed by wound healing and invasion assays. It was found that HIF-1α protein expression increases after 6 h of DMOG induction and persists for 24 h. At 6 and 24 h, a significantly higher percentage of hypoxic cells migrated compared to normoxic cells. The invasion assay demonstrated that hypoxic cells were more invasive than normoxic cells within 24 h. Thus, the increase in migration and invasion of cells is comparable to the increase in HIF-1α expression at 6 and 24 h. These findings suggest that DMOG induces HIF-1α expression in colon cancer cells, leading to enhanced cell migration and invasiveness. The established model can be further utilised in gene knockdown or drug treatment studies to evaluate the effects of hypoxia on cancer cells.

The thioredoxin system is essential for many physiological processes, including the maintenance of redox signalling pathways. Alterations in the activity, expression and interactions with other signalling pathways can lead to protective or pathophysiological responses. Thioredoxin and thioredoxin reductase, the two main components of this system, are often overexpressed in cancer, including colorectal cancer. This overexpression is often linked with tumour progression and poor outcomes. This review discusses the role of the Trx system in driving colorectal carcinogenesis and disease progression, as well as the challenges of targeting this system. Additionally, the recent advancements in the development of novel and effective thioredoxin inhibitors for colorectal cancer are also explored.

Breast cancer is a prevalent cause of global mortality, characterised by abnormal cell growth within the breast. These cells can spread to distant sites in the body through metastasis and one of the mechanisms that breast cancer cells use to metastasise is via invadopodia formation. Accumulated evidence has explained pathways that may contribute to the breast cancer cells metastasis including the ERK, SMAD-3, STAT3 and NF-κB pathways. The hypoxic conditions within tumours enhance their metastatic ability through HIF-1α upregulation. Despite advanced treatments including chemotherapy and radiotherapy, these approaches are expensive and sometimes lack efficacy. Zerumbone, a compound extracted from Zingiber zerumbet, is known for its anti-cancer properties. It counteracts cancer cell metastasis by reducing cell migration, invasion, and proliferation by acting upon multiple signalling pathways. This review recapitulates the metastasis of breast cancer and its biomarkers. In addition, our review will also explore the impact of zerumbone, therapeutic roles and its mechanism of action in reducing breast cancer metastasis.

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.

The ability to colonize distant organs which is lethal has made metastatic breast cancer become the top ten causes of mortality worldwide.Specialized actin-rich protrusions termed invadopodia were thought to be formed by highly invasive cells to degrade the extracellular matrix to drive cancer invasion and metastasis.Identification of compound(s) to hinder the formation ofinvadopodia is important to resist the metastasis of breast cancer as well as to yield antimetastasis targeted therapy.The current review aims to provide new insights on cancer invasion and candidate compound(s) capable to disrupt invadopodia formation and invadopodia-related proteins.

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.

Radiotherapy is an effective treatment for many types of cancer including breast cancer. Ionising radiation induces cytotoxicity mainly via the generation of reactive oxygen species (ROS). The overexpression of redox proteins in various tumours, including breast cancer, has been shown to cause multidrug and radioresistance. Targeting redox proteins represents an innovative approach to enhancing the radiosensitivity of tumour cells. The present study aimed to assess how inhibitors of glutathione (GSH) and thioredoxin (Trx) systems can alter the radiosensitivity of breast cancer cells. Several agents were used including Piperlongumine (PL) (targeting the GSH system), PL analogues, Indolequinone IQ9 (a novel thioredoxin reductase (TrxR) inhibitor) and metformin (a type-2 diabetes drug which inhibits the Trx system). Basal-like and luminal breast cancer cell lines were used as in vitro breast cancer models. The cytotoxic effect of drugs was assessed using growth curves and clonogenic survival assays. Clonogenic radiosensitisation was measured by treating cells with drugs and subsequently exposing them to various irradiation doses of (0-8 Gy) x-rays. The sensitiser enhancement ratio (SER) was calculated to assess the radiosensitisation effect of the drugs when combined with irradiation. Intracellular ROS levels were assessed using flow cytometry and the protein expression of Trx, thioredoxin-interacting protein (Txnip) and TrxR was assessed using Western blotting. TrxR activity was evaluated using insulin reduction assays. First, the radiosensitisation effect of PL and its analogues (LH91 and LH92) was studied in MDA-MB-231 and T47D cells. In terms of cell proliferation and clonogenic survival, MDA-MB-231 cells were more sensitive to PL, LH91 and LH92 than T47D cells (IC50 values of 3-13µM and 9-15µM, respectively). PL slightly increased the radiosensitivity of MDA-MB-231 (SER 1.12) but not that of T47D. LH91 and LH92 did not alter the breast cancer cell radioresponse. No changes in ROS levels were observed following treatment with PL, LH91 and LH92, either alone or with subsequent exposure to hydrogen peroxide (H2O2). Such results may explain the lack of a radiosensitisation effect of PL analogues in breast cancer cells. The effect of the TrxR inhibitor IQ9 in breast cancer cell lines was also investigated with respect to TrxR activity, proliferation, clonogenic survival, radioresponse and Trx system expression in MDA-MB-231, MDA-MB-468 and MDA-MB-436, T47D and MCF-7 cell lines. TrxR activity was assessed at two time points following IQ9 treatment (4 and 48 hours). IQ9 inhibited TrxR activity more efficiently in basal-like (IC50 272-439nM) than luminal (IC50 619-640nM) breast cancer cells after 4-hours of treatment. Additionally, inhibition of TrxR activity was more effective after 4-hours than 48-hours of IQ9 treatment. Cell proliferation and clonogenic survival assays revealed that IQ9 exhibited a potent anticancer effect against breast cancer cells with IC50 in the low nanomolar range (174-2072nM). Radiosensitivity of all basal-like breast cancer cells was enhanced following 4-hours of IQ9 treatment (SER 1.20-1.43), whereas a minimal or no increase in radiosensitivity was observed in MCF-7 (SER 1.08) and T47D (SER 1.00). In contrast, prolonged exposure to IQ9 (48 hours) before irradiation did not alter radiosensitivity. IQ9 treatment increased Trx expression only in MDA-MB-231 and no changes in Txnip expression were observed. IQ9 upregulated TrxR expression in luminal but not in basal-like breast cancer cells. Such results may indicate that luminal breast cancer cells increase TrxR expression to compensate for the inhibition of TrxR activity and thus reduce IQ9 radiosensitisation effects. The dual inhibition of the GSH and Trx systems using L-buthionine-S, R-sulfoximine (BSO) and IQ9 to improve the radiosensitivity of breast cancer cells was also investigated. The results show that the combined treatment of BSO and IQ9 without irradiation substantially decreased colony formation of MDA-MB-231 cells compared to the control and single agent alone. However, such effect was not observed when combined with irradiation. The final aim of the study was to evaluate whether modulating the Trx system using metformin can radiosensitise breast cancer cells and to explore whether the effect is phenotype specific. Metformin decreased breast cancer cell growth and colony formation with IC50 ranging from 3-20mM. Treatment with metformin substantially radiosensitised MCF-7 and MDA-MB-468, but not T47D cells with SER values of 1.45 and 1.30, respectively, suggesting that metformin radiosensitises breast cancer cells in a cell-specific manner. The effect of metformin may be partly explained by increasing ROS levels with subsequent exposure to H2O2. Metformin completely attenuated Txnip expression in MCF-7 and had no effect on that of T47D. MDA-MB-468 did not express Txnip. Taken together, the data indicate that the increase in radioresponse by metformin may be subject to the increase in ROS level and the loss in function of Txnip. In summary, this study highlights IQ9 and metformin as potential radiosensitisers in breast cancer via modulating Trx system function. In addition, this study provides preliminary support for the therapeutic effect of LH91, LH92, IQ9 and metformin as potential anticancer agents for the treatment of breast cancer.

Abstract 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