Hyperthermia (HT) treatment is a noninvasive cancer therapy, often used with radiation therapy and chemotherapy. Compared with 37 °C, 42 °C is mild heat stress for cells and produces reactive oxygen species (ROS) from mitochondria. To involve subsequent intracellular accumulation of DOX, we have previously reported that the expression of ATP-binding cassette sub-family G member 2 (ABCG2), an exporter of doxorubicin (DOX), was suppressed by a larger amount of intracellular mitochondrial ROS. We then hypothesized that the additive effect of HT and chemotherapy would be induced by the downregulation of ABCG2 expression via intracellular ROS increase. We used human breast cancer cell lines, MCF-7 and MDA-MB-453, incubated at 37 °C or 42 °C for 1 h to clarify this hypothesis. Intracellular ROS production after HT was detected via electron spin resonance (ESR), and DOX cytotoxicity was calculated. Additionally, ABCG2 expression in whole cells was analyzed using Western blotting. We confirmed that the ESR signal peak with HT became higher than that without HT, indicating that the intracellular ROS level was increased by HT. ABCG2 expression was downregulated by HT, and cells were injured after DOX treatment. DOX cytotoxicity enhancement with HT was considered a result of ABCG2 expression downregulation via the increase of ROS production. HT increased intracellular ROS production and downregulated ABCG2 protein expression, leading to cell damage enhancement via DOX.
Breast cancer is one of the most common types of cancers prevalent in women. Several types of breast cancers can easily metastasize to bone and cause disease complications such as hypercalcemia and pathologic fracture, thus compromising the quality of life of people affected by it. Bisphosphonate drugs are often used for the treatment of bone metastasis to suppress osteoclastic bone resorption. However, bisphosphonate has adverse effects on the gastrointestinal tract and kidneys and also induces osteonecrosis of the jaw. Photodynamic therapy (PDT) is an alternative cancer treatment approach with minimal invasiveness. It is a combination treatment that uses photosensitizers, which accumulate in tumor cells, followed by laser irradiation. We previously reported that the cellular incorporation of 5-aminolevulinic acid (5-ALA), which was a precursor of protoporphyrin IX (PpIX), was regulated by reactive oxygen species derived from mitochondria (mitROS). In this study, we investigated the incorporation of 5-ALA, accumulation of PpIX, and subsequent effects on cell viability after laser irradiation of two different breast cancer cell lines with different metastaticites. The highly metastatic breast cancer cell line 4T1E/M3 showed a significant increase in ROS production after treatment with indomethacin (IND). In addition, IND treatment enhanced the cellular uptake of 5-ALA via PEPT1 upregulation in 4T1E/M3, but not in the non-metastatic cell line. Overall, metastatic breast cancer is likely to be sensitive to ROS and activate signaling pathways associated with 5-ALA transportation, suggesting that ALA-PDT could be an effective treatment with low invasiveness for metastatic breast cancer.
Introduction: Hyperthermia (HT) is a non-invasive cancer therapy. Treatment temperature between 41°C to 44°C has no cytotoxic damage in normal cells, however shows cytotoxicity in cancer cells because of the underdeveloped vascular system. HT often used with other cancer therapy such as radiation therapy and chemotherapy. However mechanism of synergistic effect using these therapies remains unclear. Compared to 37°C, 42°C is mild heat stress for cells, thus superoxide anion is released from tissue. Superoxide anion is produced by mitochondrial electron transport chain. Reactive oxygen species (ROS), produced by mild heat stress, can be released from mitochondria. We have previously reported that ATP-binding cassette sub-family G member 2 (ABCG2) expression was suppressed by increasing mitochondrial ROS, and induction of the cancer specific porphyrin accumulation. ABCG2 is a transporter of doxorubicin (DOX), therefore we hypothesized that synergistic effect of HT and chemotherapy would be induced by down-regulation of ABCG2 expression via intracellular ROS increase. In this study, we investigated if cytotoxic effect of breast cancer cell using DOX can be enhance by HT via intracellular ROS increase. Materials and methods: The murine breast cancer cell line, 4T1E was incubated at 37°C or 42°C for 1h. Intracellular ROS generation after HT treatment was detected by electron spin resonance (ESR). Twenty four hours after HT treatment, cells were incubated in medium containing 0, 0.1 and 1 μM DOX for 24 h. Cell viability was measured using the Cell Counting Kit 8, a water-soluble tetrazolium-8 based colorimetric assay. ABCG2 expression in whole cells was analyzed by Western blotting. Results and discussion: ESR signal peak with HT treatment became high as compared to without HT treatment, indicating intracellular ROS level was increased by HT treatment. Cell viability and ABCG2 expression were decreased by DOX exposure and by HT treatment. The enhancement of HT treatment effect by DOX is considered to be result of down-regulation of ABCG2 expression by ROS. When cells were exposed to DOX with 5-aminolevulinic acid (ALA), cell viability reduced further. Since it is known that porphyrin is introduced by ALA and is transported by ABCG2, we speculate that ALA worked as a competitive inhibitor of DOX excretion transporter to enhance cell death. ESR signal peak in ALA treatment cells was higher than that in non-ALA treatment cells. Significant increase in cellular damage by HT treatment was shown by adding ALA, but not without ALA. Moreover, cell death induced by HT and ALA treatment was suppressed by adding N-acetylcysteine (NAC), which is an antioxidant. These results suggest that cellular damage of HT treatment is due to ROS production induced by ALA. Conclusion: HT treatment involved intracellular ROS production and down-regulated the expression of ABCG2 protein. HT treatment also enhanced the cell damage by DOX. Cell death by DOX was enhanced by combination with HT and ALA treatment, possibly via intracellular ROS generation, and was suppressed by additing antioxidant. Citation Format: Terasaki A, Kurokawa H, Terasaki M, Ito H, Matsui H, Ichioka E, Tsushima Y, Manaka-Iguchi A, Bando H, Hara H. Hyperthermia regulates transporter expression via ROS production and enhances the cytotoxicity of doxorubicin [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-05-05.
e18049 Background: Anaplastic thyroid carcinoma (ATC) is one of the worst prognostic malignancies with a 1-year survival rate of 5-20%. ATC is thought to be originated from differentiated thyroid carcinoma (DTC) caused by undifferentiated transformation because ATCs frequently have preexisting or coexisting region of differentiated thyroid carcinoma (co-DTC), but the detailed mechanism is unclear. In this study, we analyzed mRNA expression among ATCs, DTCs and co-DTCs, and compared their molecular characteristics. Methods: Twelve patients with ATC and eight patients with DTC, who were diagnosed at Tsukuba University Hospital between 2009 to 2020, were included in this study. Of 12 ATCs, four cases had co-DTCs. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples of ATCs, DTCs and co-DTCs. We analyzed mRNA expression profiles among three groups using nCounter PanCancer IO 360 Panel (NanoString Technologies) and nCounter Advanced Analysis (version 2.0.134). The p-values were adjusted using Benjamini-Hochberg method and adjusted p-value < 0.05 was considered as significant. Results: The 1-year survival rate of the 12 ATC cases in this study was 37.5%. Heatmap of pathway revealed that most ATCs showed up-regulation of almost all tumor-related pathways included in the panel except for autophagy while three of ATCs didn’t show immune-related pathways up-regulation, and one co-DTC sample showed similar behavior of pathways to ATCs. The top 10 up-regulated gene in co-DTCs compare to DTCs were BATF3, TDO2, PRF1, BIRC5, MKI67, ANLN, FCGR3A/B, PPARGC1B, C2 and PFKFB3, and gene set enrichment analysis revealed that the genes with differential expression were enriched in gene set of cell proliferation. The top 10 genes with differential expression in ATCs compare to co-DTCs were CXCL5, BRCA2, MMP1, CSCL6, CXCL1, DKK1 and CXCL8, for up-regulation and ID4, ARID1A and EPM2AIP1 for down-regulation. In ATCs, gene sets of cell proliferation, cytokine and chemokine signaling, and myeloid compartment were especially up-regulated. Immune cell profiling showed that ATC had abundant inflammatory cells, especially TILs, neutrophils and macrophages, while co-DTC showed intermediate characteristics between ATC and DTC. Of note, three case of ATCs had lower inflammatory cell scores than the other ATCs except for macrophages and neutrophils, and were clustered as “DTC-like”. Conclusions: These data revealed that co-DTCs showed up-regulation of cell proliferation related genes compare to DTCs, and some ATCs had different immune cell profiles. co-DTCs showed an intermediate immune cell profile between ATC and DTC. Biological profiling of ATCs, DTCs and co-DTCs may help to understand the developmental mechanism of ATCs. In this presentation, we will report the detailed results of mRNA expression analysis with discussion of the previous reports.
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