Shumei Ma

Differential roles of miR‐199a‐5p in radiation‐induced autophagy in breast cancer cells

Autophagy is a self‐degrading process that is triggered by diverse stimuli including ionizing radiation. In this study we show novel phenomena in which transfection of miR‐199a‐5p mimic significantly suppresses IR‐induced autophagy in MCF7 cells, and up‐regulates basal and IR‐induced autophagy in MDA‐MB‐231 breast cancer cells. We also identify DRAM1 and Beclin1 as novel target genes for miR‐199a‐5p. Overexpression of miR‐199a‐5p inhibits DRAM1 and Beclin1 expression in MCF7 cells, while it enhances expression of these genes in MDA‐MB‐231 cells. Furthermore, we show that miR‐199a‐5p sensitizes MDA‐MB‐231 cells to irradiation. Therefore, our data identify miR‐199a‐5p as a novel and unique regulator of autophagy, which plays an important role in cancer biology and cancer therapy.

Approach to radiation therapy in hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the 5th most common cancer and the third most common cause of cancer-related death in the world with an estimated incidence of approximately 1 million new cases annually, has becoming a major global health problem in the world. A variety of treatment modalities, including resection, liver transplantation, transarterial chemoembolization (TACE), local ablative therapy and radiation therapy (RT) have been reported. Although partial hepatectomy and liver transplantation may offer the best chance of cure, only 15% of the patients have the chance to be treated by surgery when diagnosed. The effectiveness of systemic chemotherapy for HCC has been minimal, and local ablative therapy may offer comparable survival in patients with small HCC and preserve liver function. Recently, with developments in radiotherapy techniques, radiotherapy has been shown to play potential roles in a wide spectrum of HCC and to become more important so that it is necessary to evaluate the effect of radiotherapy in treatment of HCC. This paper is aiming mainly at the current radiation therapy strategies and their current advances, the optimal radiation therapy strategies will complement the current treatments and improve the treatment efficiency.

Expression profiles of microRNAs and their target genes in papillary thyroid carcinoma.

The incidence of thyroid cancer has recently experienced a rapid increase in China, and papillary thyroid carcinoma (PTC) accounts for nearly 80% of human thyroid cancers. In the present study, the differential expression of microRNAs (miRNAs) and their target genes were identified in order to analyze the potential roles of miRNAs as biomarkers and in papillary thyroid carcinogenesis. One hundred and twenty-six PTC samples were collected from patients at the China-Japan Union Hospital, China, and the gene/miRNA expression profiles were examined with Illumina BeadChips and verified by real‑time RT-PCR. Gene Ontology (GO) categories were determined, and pathway analysis was carried out using KEGG. miRNA target genes were predicted by implementing three computational analysis programs: TargetScanS, DIANA-microT and PicTar. Two hundred and forty-eight miRNAs and 3,631 genes were found to be significantly deregulated (gene, P<0.05; miRNA, P<0.01) in PTC tissues when compared with their matching normal thyroid tissues. hsa-miR-206 (target gene, MET), hsa-miR-299-3p (target gene, ITGAV), hsa-miR-101 (target gene, ITGA3), hsa-miR-103 (target gene, ITGA2), hsa-miR‑222 (target genes, KIT and AXIN2), hsa-miR-15a (target genes, AXIN2 and FOXO1) and hsa-mir-221 (target gene, KIT) were identified. Together with the functions of the target genes, we further elucidated the role of miRNAs in papillary thyroid carcinogenesis and suggest the use of miRNAs as biomarkers for early diagnosis. Our findings provide the basis for future studies in the field of miRNA-based cancer therapy.

Low-dose radiation-induced responses: Focusing on epigenetic regulation

Purpose: With the widespread use of ionising radiation, the risks of low-dose radiation have been increasingly highlighted for special attention. This review introduces the potential role of epigenetic elements in the regulation of the effects of low-dose radiation. Materials and methods: The related literature has been analysed according to the topics of DNA methylation, histone modifications, chromatin remodelling and non-coding RNA modulation in low-dose radiation responses. Results: DNA methylation and radiation can reciprocally regulate effects, especially in the low-dose radiation area. The relationship between histone methylation and radiation mainly exists in the high-dose radiation area; histone deacetylase inhibitors show a promising application to enhance radiation sensitivity, both in the low-dose and high-dose areas; phosphorylated histone 2 AX (H2AX) shows a low sensitivity with 1–15 Gy irradiation as compared with lower dose radiation; and histone ubiquitination plays an important role in DNA damage repair mechanisms. Moreover, chromatin remodelling has an integral role in the repair of DNA double-strand breaks and the response of chromatin to ionising radiation. Finally, the effect of radiation on microRNA expression seems to vary according to cell type, radiation dose, and post-irradiation time point. Conclusion: Small advances have been made in the understanding of epigenetic regulation of low-dose radiation responses. Many questions and blind spots deserve to be investigated. Many new epigenetic elements will be identified in low-dose radiation responses, which may give new insights into the mechanisms of radiation response and their exploitation in radiotherapy.

ATM pathway is essential for ionizing radiation-induced autophagy

BACKGROUND ATM plays an important role in response to DNA damage, while the roles of ATM in radiation-induced autophagy are still unclear in cervical cancer cells. METHODS Human cervical cancer cells, Hela, were used, and cell models with ATM(-/-) and MAPK14(-/-) were established by gene engineering. Western blot was implemented to detect protein expression. MDC staining and GFP-LC3 relocalization were used to detect autophagy. CCK-8 was used to detect cell viability. Radiosensitivity was analyzed by colony formation assays. Co-immunoprecipitation was used to detect the interaction between different proteins, and apoptosis was detected by flow cytometry. RESULTS After radiation autophagy was induced, illustrated by the increase of MAPLC3-II/MAPLC3-I ratio and decrease of p62, and phosphorylation of ATM simultaneously increased. ATM(-/-) cells displayed hypersensitivity but had no influence on IR-induced apoptosis. Then inhibitor of ATM, KU55933, ATM and MAPK14 silencing were used, and autophagy was induced by IR more than 200% in control, and only by 35.72%, 53.18% and 24.76% in KU55933-treated cells, ATM(-/-) and MAPK14(-/-) cells, respectively. KU55933 inhibited IR-induced autophagy by activating mTOR pathways. ATM silencing decreased the expression of MAPK14 and mTOR signals significantly. Beclins bond to PI3KIII and their interaction increased after IR, while in ATM(-/-) and MAPK14(-/-) cells this interaction decreased after IR. Both ATM and MAPK14 interacted with Beclin, while ATM(-/-) and MAPK14(-/-) cells showed no interaction. CONCLUSIONS ATM could promote IR-induced autophagy via the MAPK14 pathway, the mTOR pathway, and Beclin/PI3KIII complexes, which contributed to the effect of ATM on radiosensitivity.

MicroRNA-18a upregulates autophagy and ataxia telangiectasia mutated gene expression in HCT116 colon cancer cells

Autophagy is an evolutionarily conserved, multi-step lysosomal degradation process in which a cell degrades its own long-lived proteins and damaged organelles. Ataxia telangiectasia mutated (ATM) has recently been shown to upregulate the process of autophagy. Previous studies showed that certain microRNAs, including miR-18a, potentially regulate ATM in cancer cells. However, the mechanisms behind the modulation of ATM by miR-18a remain to be elucidated in colon cancer cells. In the present study, we explored the impact of miR-18a on the autophagy process and ATM expression in HCT116 colon cancer cells. To determine whether a preliminary link exists between autophagy and miR-18a, HCT116 cells were irradiated and quantitative (q) PCR was performed to measure miR-18a expression. HCT116 cells were transfected with an miR-18a mimic to study its impact on indicators of autophagy. Western blotting and luciferase assays were implemented to explore the impact of miR-18a on ATM gene expression in HCT116 cells. The results showed that miR-18a expression was strongly stimulated by radiation. Ectopic overexpression of miR-18a in HCT116 cell lines potently enhanced autophagy and ionizing radiation-induced autophagy. Moreover, miR-18a overexpression led to the upregulation of ATM expression and suppression of mTORC1 activity. Results of the present study pertaining to the role of miR-18a in regulating autophagy and ATM gene expression in colon cancer cells revealed a novel function for miR-18a in a critical cellular event and on a crucial gene with significant impacts in cancer development, progression, treatment and in other diseases.

The Tumor Suppressor, p53, Contributes to Radiosensitivity of Lung Cancer Cells by Regulating Autophagy and Apoptosis

PURPOSE Cell death is one of the most important endpoints of radiosensitivity. The tumor suppressor p53 participates not only in regulation of apoptosis, but also in autophagy mechanism. In this study, H1299-P53 (with wild-type p53) and H1299-175H (with mutant 175H) were used, and the effects of p53 on radiosensitivity were analyzed. METHODS Cell models with different p53 status were established by gene engineering, and cell viability was examined by colony formation assay, and cell counting kit-8 (CCK-8), 3-Methyladenine, and Z-VAD were used to block autophagy and apoptosis, respectively. Western blot was used to detect protein expression; monodansylcadaverine (MDC) staining was used to analyze autophagy rate; DAPI/Propidium Iodide (PI) staining and flow cytometry were used to assess apoptosis and necrosis. RESULTS In parental H1299, H1299-P53, and H1299-175H cells, radiosensitivity exhibited different by colony formation and CCK-8 assay (D0: 1.764 Gy, 1.407 Gy and 1.695 Gy; Dq: 2.977 Gy, 1.199 Gy and 2.312 Gy in turn). The radiosensitization of p53 was associated with the increase of MDM2 and P21 expression. The ionizing radiation (IR)-induced apoptosis was significant in H1299-P53 compared with in H1299 and H199-175H (p < 0.05) by flow cytometry, and the expression of cleaved-caspase3 was increased in H1299-P53 cells. While the IR-induced autophagy was significant in H1299 cells (p < 0.01) and decreased in H1299-P53 and H1299-175H cells (p < 0.01) by MDC staining, the expression of MAPLC3II and Beclin-1 increased in H1299, but not in H1299-p53 and H199-175H cells. The IR-induced cell survival was significantly increased by Z-VAD-FMK and decreased by 3MA in H1299-P53 cells; IR- induced autophagy was significantly increased by Z-VAD-FMK in H1299-P53 cells (p < 0.01), but not changed in H1299 cells. CONCLUSION p53 could regulate radiosensitivity by inhibiting autophagy and activating apoptosis; autophagy provides a prosurvival mechanism, and p53 potently abrogated the IR-induced autophagy, while mutant 175H shown no effect on radiosensitivity, suggesting that individual treatment strategies should be based on p53 status in patients.

Autophagy inhibition plays the synergetic killing roles with radiation in the multi-drug resistant SKVCR ovarian cancer cells

PurposeAutophagy has attracted attentions as a novel mechanism for tumor development. In this study Human ovarian carcinoma cell line SKOV3 and multidrug-resistant phenotype SKVCR cells were used and the roles of autophagy in radiation-induced cell death were analyzed.Methods and materialsCell viability was examined by colony formation and cell counting kit-8 (CCK-8) assay, 3MA and ZVAD were used to block autophagy and apoptosis, respectively. Quantitative real-time PCR was used to detect mRNA level and Western blot was used to detect protein expression, monodansylcadaverine (MDC) staining and flow cytometery were used for autophagy, apoptosis and cell cycle dynamics, respectively.Results(1) The radiosensitivity exhibited differently in SKOV3 and SKVCR cells (SKOV3: D0=3.37, SKVCR: D0= 4.18); compared with SKOV3 the constitutive expression of MAPLC3 in SKVCR was higher, but no change of Caspase-3 and cleaved Caspase-3. (2) The ionizing radiation (IR)- induced apoptosis and autophagy were significant in both cells (P<0.05); inhibition of apoptosis with ZVAD showed no impact on survival of SKOV3 and SKVCR cells after radiation, while inhibition of autophagy significantly decreased viability in SKVCR cells, for SKVO3 cells only low level of radiation (2 Gy and 4 Gy) could decrease the viability(P<0.05). (3) ZVAD inhibited apoptosis and autophagy in both cells, 3MA inhibit apoptosis in SKOV3, and promote apoptosis in SKVCR, together with inhibition of autophagy. (4) G2/M arrest was induced by radiation in both cells; the accumulation of G2/M was more significant in SKOV3, 3MA attenuated the radiation-induced S phase delay in SKVCR.ConclusionIR-induced autophagy provides a self-protective mechanism against radiotherapy in SKVCR cells, the use of autophagy inhibitor, 3MA, increases the killing effects of radiation by inhibiting autophagy and radiation- induced S phase delay, also by the increase of apoptosis, which suggests a better therapeutic strategy in drug- resistant SKVCR ovarian cancer cells.

Synergistic killing of lung cancer cells by cisplatin and radiation via autophagy and apoptosis

Cisplatin is a commonly used drug for chemotherapy, however, whether it may be used synergistically with radiotherapy remains unclear. The present study investigated the underlying mechanisms of synergistic killing by radiosensitization and cisplatin, with a focus on the growth inhibition, apoptosis and autophagy of non-small cell human lung cancer cells in vitro and in a tumor xenograft in vivo. A549 cells were used for the in vitro experiments and divided into the following four treatment groups: Sham-irradiated; conventional radiotherapy (CRT) of five doses of 2 Gy every day; hyperfractionated radiotherapy of five doses of 2 Gy (1 Gy twice a day at 4 h intervals) every day; and CRT plus cisplatin. A xenograft tumor-bearing C57BL/6 model was established for the in vivo experiments and the above-mentioned treatments were administered. MTT and colony formation assays were used to detect cell viability and western blotting was performed to detect the levels of protein expression. Monodansylcadaverine staining and the immunofluorescence technique were used to analyze the autophagy rate, while flow cytometry and immunohistochemistry were performed to detect the expression levels of the genes associated with apoptosis and autophagy, including microtubule-associated protein 1 light chain 3 (MAPLC3)-II, phosphoinositide 3-kinase (PI3K) III, Beclin1, phosphorylated protein kinase B (p-AKT), damage-regulated autophagy modulator (DRAM), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein, caspase-3 and p21. The MTT assay demonstrated that cisplatin exhibits a dose-dependent cytotoxicity in A549 cells and synergizes with radiation to promote the cell-killing effect of radiation. In the xenograft mouse model of Lewis cells, cisplatin plus ionizing radiation (IR) (five doses of 2 Gy) yielded the most significant tumor suppression. The autophagic vacuoles, the ratio of MAPLC3-II to MAPLC3-I (LC3-II/LC3-I) and the levels of Beclin1 were found to increase in all treatment groups, with the most marked upregulation observed in the CRT plus cisplatin treatment group. In addition, caspase-3 processing was enhanced in the group treated with the combination of cisplatin with radiation, compared with the group treated with radiation alone. Fractionated IR resulted in a significant increase in p21 expression, which was further enhanced when combined with cisplatin. Furthermore, treatment with cisplatin and fractionated IR resulted in a significant elevation of the expression of the autophagy-related genes, PI3KIII, Beclin1 and DRAM1. However, the levels of p-AKT were observed to decline following exposure to fractionated IR in the presence or absence of cisplatin. As for the apoptosis signaling genes, the combination of cisplatin and fractionated IR therapy resulted in a significant decrease in Bcl-2 expression and a marked upregulation of p21 expression. The current study offers strong evidence that the combination of cisplatin with radiation strengthens the killing effect of radiation via pro-apoptotic and pro-autophagic cell death.

The synergistic effects of traditional Chinese herbs and radiotherapy for cancer treatment (Review)

Traditional Chinese medicine (TCM) has been demonstrated to have potent cytotoxic activity against certain malignant tumors. Ionizing radiation (IR) is one of the most effective methods used in the clinical treatment of cancer. The drawback of a single formula is that it limits the treatment efficacy for cancer, while comprehensive strategies require additional theoretical support. However, a combination of different antitumor treatment modalities is advantageous in restricting the non-specific toxicity often observed with an extremely high dose of a single regimen. The induction of apoptotic cell death is a significant process in tumor cells following radiotherapy or chemotherapy, and resistance to these treatments has been linked to a low propensity for apoptosis. Autophagy is a response of cancer cells to IR or chemotherapy, and involves the prominent formation of autophagic vacuoles in the cytoplasm. In this review, the synergistic effects of TCM and radiotherapy are summarized and the underlying mechanisms are illustrated, providing new therapeutic strategies for cancer.