Orthogonal Optimization, Characterization, and In Vitro Anticancer Activity Evaluation of a Hydrogen Peroxide-Responsive and Oxygen-Reserving Nanoemulsion for Hypoxic Tumor Photodynamic Therapy.

Tumor hypoxia can seriously impede the effectiveness of photodynamic therapy (PDT). To address this issue, two approaches, termed in situ oxygen generation and oxygen delivery, were developed. The in situ oxygen generation method uses catalysts such as catalase to decompose excess H2O2 produced by tumors. It offers specificity for tumors, but its effectiveness is limited by the low H2O2 concentration often present in tumors. The oxygen delivery strategy relies on the high oxygen solubility of perfluorocarbon, etc., to transport oxygen. It is effective, but lacks tumor specificity. In an effort to integrate the merits of the two approaches, we designed a multifunctional nanoemulsion system named CCIPN and prepared it using a sonication-phase inversion composition-sonication method with orthogonal optimization. CCIPN included catalase, the methyl ester of 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me), photosensitizer IR780, and perfluoropolyether. Perfluoropolyether may reserve the oxygen generated by catalase within the same nanoformulation for PDT. CCIPN contained spherical droplets below 100 nm and showed reasonable cytocompatibility. It presented a stronger ability to generate cytotoxic reactive oxygen species and consequently destroy tumor cells upon light irradiation, in comparison with its counterpart without catalase or perfluoropolyether. This study contributes to the design and preparation of oxygen-supplementing PDT nanomaterials.

Isoliquiritigenin Nanoemulsion Preparation by Combined Sonication and Phase-Inversion Composition Method: In Vitro Anticancer Activities.

Isoliquiritigenin (ILQ) has a number of biological activities such as antitumor and anti-inflammatory effects. However, biomedical applications of ILQ are impeded by its poor aqueous solubility. Therefore, in this research, we prepared a novel ILQ-loaded nanoemulsion, i.e., ILQ-NE, which consisted of Labrafil® M 1944 CS (oil), Cremophor® EL (surfactant), ILQ, and phosphate-buffered saline, by employing a combined sonication (high-energy) and phase-inversion composition (low-energy) method (denoted as the SPIC method). The ILQ-NE increased the ILQ solubility ~1000 times more than its intrinsic solubility. It contained spherical droplets with a mean diameter of 44.10 ± 0.28 nm and a narrow size distribution. The ILQ loading capacity was 4%. The droplet size of ILQ-NE remained unchanged during storage at 4 °C for 56 days. Nanoemulsion encapsulation effectively prevented ILQ from degradation under ultraviolet light irradiation, and enhanced the ILQ in vitro release rate. In addition, ILQ-NE showed higher cellular uptake and superior cytotoxicity to 4T1 cancer cells compared with free ILQ formulations. In conclusion, ILQ-NE may facilitate the biomedical application of ILQ, and the SPIC method presents an attractive avenue for bridging the merits and eliminating the shortcomings of traditional high-energy methods and low-energy methods.

Recent Advances in Strategies for Addressing Hypoxia in Tumor Photodynamic Therapy.

Photodynamic therapy (PDT) is a treatment modality that uses light to target tumors and minimize damage to normal tissues. It offers advantages including high spatiotemporal selectivity, low side effects, and maximal preservation of tissue functions. However, the PDT efficiency is severely impeded by the hypoxic feature of tumors. Moreover, hypoxia may promote tumor metastasis and tumor resistance to multiple therapies. Therefore, addressing tumor hypoxia to improve PDT efficacy has been the focus of antitumor treatment, and research on this theme is continuously emerging. In this review, we summarize state-of-the-art advances in strategies for overcoming hypoxia in tumor PDTs, categorizing them into oxygen-independent phototherapy, oxygen-economizing PDT, and oxygen-supplementing PDT. Moreover, we highlight strategies possessing intriguing advantages such as exceedingly high PDT efficiency and high novelty, analyze the strengths and shortcomings of different methods, and envision the opportunities and challenges for future research.

Involvement of twist in NNK exposure-promoted lung cancer cell migration and invasion.

Nicotine-derived nitrosamine ketone (NNK), one of the potent carcinogens in cigarette smoke, has been reported to facilitate lung cancer cell migration and invasion. Twist plays an important role in regulating migration and invasion of lung cancer cells. However, it is unclear whether Twist is implicated in NNK-induced migration and invasion of lung cancer cells. Lung cancer cells were exposed to various doses of NNK for four weeks. The expression levels of protein and mRNA were detected by western blot and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Small interfering RNA (siRNA) was applied to knock down the expression of Twist. The ability of cell migration and invasion was evaluated by wound-healing assay and Transwell invasion assay. NNK exposure increased the levels of Twist protein and mRNA expression in lung cancer cells compared to solvent control. Lung cancer cells exposed to NNK exhibited higher ability of migration and invasion than those with solvent control did. Twist silencing could block NNK-promoted migration and invasion of lung cancer cells. NNK exposure increased the expression levels of N-cadherin mRNA and decreased the expression levels of E-cadherin mRNA in lung cancer cells, which could be modulated by Twist silencing. In conclusion, Twist was involved in NNK-induced migration and invasion of lung cancer cells.

Involvement of p38 MAPK pathway in benzo(a)pyrene-induced human hepatoma cell migration and invasion.

The objective of this study was to investigate the potential role of p38 mitogen-activated protein kinases (MAPK) in benzo(a)pyrene (BaP)-induced hepatoma cell migration and invasion. Western blot assay was applied to detect the expression of proteins. qRT-PCR assay was used to measure the expression of mRNA. Wound healing assay and Transwell invasion assay were performed to evaluate cell migratory ability and cell invasive ability, respectively. Our data showed that BaP exposure increased the expression of p-p38 protein in human hepatoma HepG2 cells. Exposure to BaP facilitated HepG2 cell migration and invasion, which could be blocked by p38 MAPK inhibitors. In addition, BaP exposure induced upregulation of MMP9 mRNA expression, which was modulated by p-p38. In conclusion, p38 MAPK pathway was involved in BaP-induced hepatoma cell migration and invasion.

The roles of TG-interacting factor in cadmium exposure-promoted invasion and migration of lung cancer cells.

Cadmium (Cd) is an established human carcinogen. Previous documents have demonstrated the association of Cd exposure with lung cancer progression. However, the underlying mechanisms of Cd-promoted invasion and migration of lung cancer cells have not yet been fully understood. The objective of this study was to explore the functional roles of TG-interacting factor (TGIF) in Cd exposure-promoted invasion and migration of lung cancer cells. Lung cancer cells were exposed to Cd for eight weeks. Invasion assay and migration assay were used to evaluate the cell invasive and migratory ability. qRT-PCR and western blot analyses were applied to analyze the expression levels of mRNA and protein. Our results showed that Cd-exposed lung cancer cells expressed markedly increased TGIF protein and mRNA compared to control cells. Cd-exposed lung cancer cells exhibited higher migratory and invasive ability than control cells did. However, silencing TGIF blocked Cd exposure-accelerated lung cancer cell invasion and migration. In addition, we found that the upregulation of Snail protein expression induced by exposure to Cd was modulated by TGIF in lung cancer cells. In conclusion, our data demonstrated that TGIF might play a crucial role in invasion and migration of lung cancer cells exposed to Cd.

Long-term cadmium exposure promoted breast cancer cell migration and invasion by up-regulating TGIF.

Cadmium (Cd) is a known human carcinogen. Previous studies have demonstrated that Cd exposure promoted migration and invasion of breast cancer cells. However, the molecular mechanisms underlying this process have not yet been clearly addressed. The purpose of this study was to investigate whether TG-interacting factor (TGIF) was involved in long-term Cd exposure-induced migration and invasion of breast cancer cells. Human breast cancer cells were continuously exposed to Cd for eight weeks. Western blot and qRT-PCR assays were performed to measure the expression of protein and mRNA. Migration and invasion assays were performed to assess the migratory and invasive ability of human breast cancer cells. Our data indicated that long-term Cd exposure obviously increased the expression of TGIF protein and mRNA in human breast cancer cells. Long-term Cd exposure increased the ability of migration and invasion of human breast cancer cells, which could be inhibited by transfection of small interfering RNA (siRNA) targeting TGIF. We also observed that the long-term Cd exposure-induced up-regulation of MMP2 mRNA expression was modulated by TGIF. In conclusion, our findings suggested that TGIF/MMP2 signaling axis might be involved in malignant progression stimulated by long-term Cd exposure in human breast cancer.

Silencing TGIF suppresses migration, invasion and metastasis of MDA­MB­231 human breast cancer cells.

This study explored the potential role of TG-interacting factor (TGIF) in migration, invasion and metastasis of the human breast cancer cell line MDA­MB­231. Western blot assay, immunohistochemistry and qRT-PCR assays were applied to detect the expression of protein and mRNA. Wound healing assay, Transwell invasion assay and tail vein metastatic assay were performed to assess the migration, invasion and metastasis of stable TGIF-silenced MDA­MB­231 cell line in vitro and in vivo. The significantly higher frequency of TGIF high-expression was observed in metastatic breast cancer (62.9%) compared to that in non-metastatic breast cancer (25.8%). Silencing TGIF suppressed migration and invasion of MDA­MB­231 cells in vitro and tumor metastasis in nude mouse models. The expression of Snail1, matrix metalloproteinase 2 (MMP2) and ß-catenin was markedly decreased in the stable TGIF-silenced MDA­MB­231 cells compared with the control cells. Our results suggest that silencing TGIF suppressed the migration, invasion and metastasis of the human breast cancer cell line MDA­MB­231 using in vitro and in vivo experiments.

Overexpression of SIRT6 attenuates the tumorigenicity of hepatocellular carcinoma cells.

OBJECTIVE: This study aimed to explore the effects of overexpression of sirtuin 6 (SIRT6) on the tumorigenicity of hepatocellular carcinoma (HCC) cells. METHODS: Stable SIRT6-overexpressed HCC cell lines were established by transfecting SIRT6 plasmid. Soft agar assay and tumor xenograft assay in nude mice were applied. Flow cytometry was employed to detect cell cycle distribution. Western blotting analysis was used to detect the expression of proteins. RESULTS: Overexpression of SIRT6 attenuated HepG2 and HCCLM3 cells proliferation, colony formation in vitro and tumor formation in nude mice, and resulted in the G1 phase cell cycle arrest. Overexpression of SIRT6 reduced the expression of cyclin D1 and p-ERK proteins in both HepG2 and HCCLM3 cells. CONCLUSION: Overexpression of SIRT6 attenuates the tumorigenicity of HCC cells.

STIM1 silencing inhibits the migration and invasion of A549 cells.

The present study aimed to explore the effects of stromal interaction molecule 1 (STIM1) knockdown on the migration, invasion and metastasis of A549 cells in vitro and in vivo. Western blotting and immunohistochemistry were used to detect protein expression levels. Wound healing and Transwell invasion assays were used to assess the migratory and invasive abilities of A549 cells transfected with STIM1­specific short hairpin (sh)RNA (shSTIM1). In addition, a tail vein metastatic assay was performed. The results demonstrated that the frequency of STIM1 high­expression was significantly increased in metastatic lung cancer tissues (72.2%) compared with in non­metastatic lung cancer tissues (33.0%). STIM1 knockdown inhibited A549 cell migration and invasion in vitro and tumor metastasis in vivo. The protein expression levels of Snail1, Vimentin, matrix metalloproteinase (MMP) 2 and MMP9 were markedly decreased in A549­shSTIM1 compared with in A549 cells transfected with control shRNA (shcon). In addition, the protein expression levels of E­cadherin were markedly increased in A549­shSTIM1 cells compared with in A549­shcon cells. These results suggested that STIM1 knockdown may inhibit the migration and invasion of A549 cells in vitro, and metastasis in vivo.

Knockdown of TGIF attenuates the proliferation and tumorigenicity of EC109 cells and promotes cisplatin-induced apoptosis.

A previous study has reported that frequent amplifications of the TG-interacting factor (TGIF) were observed in esophageal squamous cell carcinoma. The aim of the present study was to investigate the potential role of TGIF in the proliferation and tumorigenicity of the esophageal cancer cell line EC109 and cisplatin-induced apoptosis. Stable TGIF-knockdown EC109 cell line was established by infecting short hairpin RNA (shRNA) lentiviral particles. Soft agar and tumor xenograft assays were applied in nude mice. Flow cytometry was employed to evaluate the cell cycle and apoptosis. Western blot analysis was used to detect the expression of proteins. TGIF knockdown suppressed EC109 cell proliferation, colony formation in soft agar and tumor growth in nude mice, induced cell cycle arrest in the G1 phase, and promoted cisplatin-induced apoptosis. In addition, TGIF knockdown significantly reduced the expression of phospho-Rb in EC109 cells. The reduced level of full length PARP expression and the increased level of cleaved caspase-3 expression were observed in EC109 cells with the treatment of cisplatin and TGIF knockdown. The results suggest that knockdown of TGIF attenuated the proliferation and tumorigenicity of EC109 cells, and promoted cisplatin-induced apoptosis.

Elevated expression of STIM1 is involved in lung tumorigenesis.

This study aimed to address the potential role of STIM1 (stromal interaction molecule 1) in lung tumorigenesis. Colony formation in soft agar assay and tumorigenicity in nude mice assay were conducted. Western blot, immunohistochemistry and quantitative real-time polymerase chain reaction were used to measure the STIM1 expression. The distribution of cell cycle was detected by flow cytometry assay. Our results showed that the expression of STIM1 mRNA was significantly higher in human lung tumors than that in adjacent non-neoplastic lung tissues. Significantly increased expression of STIM1 mRNA and protein was observed in 16HBE-benzo(a)pyrene (BaP) cells and in BaP-treated mice lung tissues compared with 16HBE-control cells and the control group, respectively. Silencing STIM1 inhibited the proliferation and colony formation of A549 cells in in vitro experiments, attenuated the growth of tumor xenografts of A549 cells in in vivo experiments and induced the arrest of cell cycle in the G1 phase. The markedly decreased expression of cyclin D1 protein was observed in A549-shRNA-STIM1 cells as compared to A549-shRNA-control cells. The markedly increased expression of p21 protein was observed in A549-shRNA-STIM1 cells as compared to A549-shRNA-control cells. The expression levels of ß-catenin and TGIF proteins were lower in A549-shRNA-STIM1 cells than those in A549-shRNA-control cells. In conclusion, this study indicated that the elevated expression of STIM1 might be involved in lung tumorigenesis.

Overexpression of PHRF1 attenuates the proliferation and tumorigenicity of non-small cell lung cancer cells.

The aim of this study was to investigate the potential role of PHRF1 in lung tumorigenesis. Western blot analysis was used to detect the expression of proteins. Quantitative reverse transcriptase polymerase chain reaction, immunohistochemistry, soft agar assay and tumor formation assay in nude mice were applied. Cell cycle distribution was analyzed by flow cytometry. The lower level of PHRF1 mRNA was observed in human lung cancer tissues than that in paracancerous tissues. The decreased expression of PHRF1 protein was observed in H1299 and H1650 cell lines than that in 16HBE and BEAS-2B cell lines. The decreased expression of PHRF1 protein was observed in malignant 16HBE cells compared to control cells. The reduced expression of PHRF1 protein was observed in mice lung tissues treated with BaP than that in control group. Overexpression of PHRF1 inhibited H1299 cell proliferation, colony formation in vitro and growth of tumor xenograft in vivo, and arrested cell cycle in G1 phase. The decreased expression of TGIF and c-Myc proteins and the increased expression of p21 protein were observed in H1299-PHRF1 cells compared with H1299-pvoid cells. In conclusion, our findings suggest that overexpression of PHRF1 attenuated the proliferation and tumorigenicity of non-small cell lung cancer cell line of H1299.

Silencing of TGIF attenuates the tumorigenicity of A549 cells in vitro and in vivo.

The aim of this study was to investigate the effects of the silencing of the TG-interacting factor (TGIF) on the tumorigenicity of A549 cells in vitro and in vivo. Stable TGIF-silenced A549 cells were established by infecting shRNA lentiviral particles. Western blotting analysis was used to detect the expression of proteins. Cell cycle was detected by flow cytometry. Soft agar assay and tumor formation assay in nude mice were applied. The silencing of TGIF inhibited A549 cell proliferation, colony formation in vitro, growth of tumor xenograft in vivo, and arrested the cell cycle in the G1 phase. The expression of CDK4, cyclin D1, and phospho-Rb was markedly decreased in the A549-shTGIF cells compared with the A549-shcon cells, and p21 was markedly increased in the A549-shTGIF cells compared with the A549-shcon cells. A lower level of ß-Catenin protein expression was observed in the A549-shTGIF cells than that in the A549-shcon cells. The silencing of TGIF attenuates the tumorigenicity of A549 cells in vitro and in vivo.

Elevated expression of TGIF is involved in lung carcinogenesis.

The purpose of this study was to explore the expression of TG-interacting factor (TGIF) in lung carcinogenesis. Malignant transformation of human bronchial epithelial (16HBE) cell was established by benzo(a)pyrene (BaP) treatment. Soft agar assay and tumor formation assay in nude mice were applied. Tumorigenesis experiment in vivo was done by BaP treatment. Western blotting, immunohistochemistry, and quantitative polymerase chain reaction were used to detect TGIF expression. We observed a higher level of TGIF messenger RNA (mRNA) in lung cancer tissues than that in paracancerous tissues. We observed significantly higher levels of TGIF mRNA and protein in A549 and H1299 cell lines than that in 16HBE cell. Increased expressions of TGIF protein and mRNA were observed in 16HBE cells induced by BaP treatment as compared to those in solvent control group. We observed significantly higher levels of TGIF mRNA and protein in 16HBE-BaP cells than that in 16HBE-control cells. We observed significantly higher levels of TGIF mRNA and protein in mice lung tissues treated with BaP than that in control group. Our results suggested that elevated expression of TGIF was involved in lung carcinogenesis.