Yu-Ying He

Enhanced Photodynamic Efficacy towards Melanoma Cells by Encapsulation of Pc4 in Silica Nanoparticles

Nanoparticles have been explored recently as an efficient means of delivering photosensitizers for cancer diagnosis and photodynamic therapy (PDT). Silicon phthalocyanine 4 (Pc4) is currently being clinically tested as a photosensitizer for PDT. Unfortunately, Pc4 aggregates in aqueous solutions, which dramatically reduces its PDT efficacy and therefore limits its clinical application. We have encapsulated Pc4 using silica nanoparticles (Pc4SNP), which not only improved the aqueous solubility, stability, and delivery of the photodynamic drug but also increased its photodynamic efficacy compared to free Pc4 molecules. Pc4SNP generated photo-induced singlet oxygen more efficiently than free Pc4 as measured by chemical probe and EPR trapping techniques. Transmission electron microscopy and dynamic light scattering measurements showed that the size of the particles is in the range of 25-30 nm. Cell viability measurements demonstrated that Pc4SNP was more phototoxic to A375 or B16-F10 melanoma cells than free Pc4. Pc4SNP photodamaged melanoma cells primarily through apoptosis. Irradiation of A375 cells in the presence of Pc4SNP resulted in a significant increase in intracellular protein-derived peroxides, suggesting a Type II (singlet oxygen) mechanism for phototoxicity. More Pc4SNP than free Pc4 was localized in the mitochondria and lysosomes. Our results show that these stable, monodispersed silica nanoparticles may be an effective new formulation for Pc4 in its preclinical and clinical studies. We expect that modifying the surface of silicon nanoparticles encapsulating the photosensitizers with antibodies specific to melanoma cells will lead to even better early diagnosis and targeted treatment of melanoma in the future.

Regulation of global genome nucleotide excision repair by SIRT1 through xeroderma pigmentosum C

Disruption of the nucleotide excision repair (NER) pathway by mutations can cause xeroderma pigmentosum, a syndrome predisposing affected individuals to development of skin cancer. The xeroderma pigmentosum C (XPC) protein is essential for initiating global genome NER by recognizing the DNA lesion and recruiting downstream factors. Here we show that inhibition of the deacetylase and longevity factor SIRT1 impairs global genome NER through suppressing the transcription of XPC in a SIRT1 deacetylase-dependent manner. SIRT1 enhances XPC expression by reducing AKT-dependent nuclear localization of the transcription repressor of XPC. Finally, we show that SIRT1 levels are significantly reduced in human skin tumors from Caucasian patients, a population at highest risk. These findings suggest that SIRT1 acts as a tumor suppressor through its role in DNA repair.

Regulation of cell proliferation and migration by p62 through stabilization of Twist1

Significance The selective autophagy substrate p62 has been shown to regulate inflammatory and redox pathways in tumorigenesis following autophagy suppression. Here we have elucidated the critical role of p62 in promoting cell proliferation and migration in vitro and tumor growth and metastasis in vivo through its molecular interaction with the oncogenic transcription factor Twist1. Our findings suggest that targeting the interaction between p62 and Twist1 has potential for cancer prevention and therapy. The selective autophagy substrate p62 serves as a molecular link between autophagy and cancer. Suppression of autophagy causes p62 accumulation and thereby contributes to tumorigenesis. Here we demonstrate that autophagy deficiency promotes cell proliferation and migration through p62-dependent stabilization of the oncogenic transcription factor Twist1. p62 binds to Twist1 and inhibits degradation of Twist1. In mice, p62 up-regulation promotes tumor cell growth and metastasis in a Twist1-dependent manner. Our findings demonstrate that Twist1 is a key downstream effector of p62 in regulation of cell proliferation and migration and suggest that targeting p62-mediated Twist1 stabilization is a promising therapeutic strategy for prevention and treatment of cancer.

Recent advances in the prevention and treatment of skin cancer using photodynamic therapy.

Photodynamic therapy (PDT) is a noninvasive procedure that involves a photosensitizing drug and its subsequent activation by light to produce reactive oxygen species that specifically destroy target cells. Recently, PDT has been widely used in treating non-melanoma skin malignancies, the most common cancer in the USA, with superior cosmetic outcomes compared with conventional therapies. The topical ‘photosensitizers’ commonly used are 5-aminolevulinic acid (ALA) and its esterified derivative methyl 5-aminolevulinate, which are precursors of the endogenous photosensitizer protoporphyrin IX. After treatment with ALA or methyl 5-aminolevulinate, protoporphyrin IX preferentially accumulates in the lesion area of various skin diseases, which allows not only PDT treatment but also fluorescence diagnosis with ALA-induced porphyrins. Susceptible lesions include various forms of non-melanoma skin cancer such as actinic keratosis, basal cell carcinoma and squamous cell carcinoma. The most recent and promising developments in PDT include the discovery of new photosensitizers, the exploitation of new drug delivery systems and the combination of other modalities, which will all contribute to increasing PDT therapeutic efficacy and improving outcome. This article summarizes the main principles of PDT and its current clinical use in the management of non-melanoma skin cancers, as well as recent developments and possible future research directions.

PTEN in DNA damage repair

The ability of DNA repair in a cell is vital to its genomic integrity and thus to the normal functioning of an organism. Phosphatase and tensin homolog (PTEN) is a well-established tumor suppressor gene that induces apoptosis and controls cell growth by inhibiting the PI3K/AKT pathway. In various human cancers, PTEN is frequently found to be mutated, deleted, or epigenetically silenced. Recent new findings have demonstrated that PTEN also plays a critical role in DNA damage repair and DNA damage response. This review summarizes the recent progress in the function of PTEN in DNA damage repair, especially in double strand break repair and nucleotide excision repair. In addition, we will discuss the role of PTEN in DNA damage response through its interaction with the Chk1 and p53 pathways. We will focus on the newly discovered mechanisms and the potential implications in cancer prevention and therapeutic intervention.

Targeting the AMP-Activated Protein Kinase for Cancer Prevention and Therapy

Despite the advances in biomedical research and clinical applications, cancer remains a leading cause of death worldwide. Given the limitations of conventional chemotherapeutics, including serious toxicities and reduced quality of life for patients, the development of safe and efficacious alternatives with known mechanism of action is much needed. Prevention of cancer through dietary intervention may hold promise and has been investigated extensively in the recent years. AMP-activated protein kinase (AMPK) is an energy sensor that plays a key role in the regulation of protein and lipid metabolism in response to changes in fuel availability. When activated, AMPK promotes energy-producing catabolic pathways while inhibiting anabolic pathways, such as cell growth and proliferation – thereby antagonizing carcinogenesis. Other anti-cancer effects of AMPK may include promoting autophagy and DNA repair upon UVB damage. In the last decade, interest in AMPK has grown extensively as it emerged as an attractive target molecule for cancer prevention and treatment. Among the latest developments is the activation of AMPK by naturally occurring dietary constituents and plant products – termed phytochemicals. Owing to their efficacy and safety, phytochemicals are considered as an alternative to the conventional harmful chemotherapy. The rising popularity of using phytochemicals for cancer prevention and therapy is supported by a substantial progress in identifying the molecular pathways involved, including AMPK. In this article, we review the recent progress in this budding field that suggests AMPK as a new molecular target in the prevention and treatment of cancer by phytochemicals.

Pristine (C60) and hydroxylated [C60(OH)24] fullerene phototoxicity towards HaCaT keratinocytes: type I vs type II mechanisms.

The increasing use of fullerene nanomaterials has prompted widespread concern over their biological effects. Herein, we have studied the phototoxicity of gamma-cyclodextrin bicapped pristine C 60 [(gamma-CyD) 2/C 60] and its water-soluble derivative C 60(OH) 24 toward human keratinocytes. Our results demonstrated that irradiation of (gamma-CyD) 2/C 60 or C 60(OH) 24 in D 2O generated singlet oxygen with quantum yields of 0.76 and 0.08, respectively. Irradiation (>400 nm) of C 60(OH) 24 generated superoxide as detected by the EPR spin trapping technique; superoxide generation was enhanced by addition of the electron donor nicotinamide adenine dinucleotide (reduced) (NADH). During the irradiation of (gamma-CyD) 2/C 60, superoxide was generated only in the presence of NADH. Cell viability measurements demonstrated that (gamma-CyD) 2/C 60 was about 60 times more phototoxic to human keratinocytes than C 60(OH) 24. UVA irradiation of human keratinocytes in the presence of (gamma-CyD) 2/C 60 resulted in a significant rise in intracellular protein-derived peroxides, suggesting a type II mechanism for phototoxicity. UVA irradiation of human keratinocytes in the presence of C 60(OH) 24 produced diffuse intracellular fluorescence when the hydrogen peroxide probe Peroxyfluor-1 was present, suggesting a type I mechanism. Our results clearly show that the phototoxicity induced by (gamma-CyD) 2/C 60 is mainly mediated by singlet oxygen with a minor contribution from superoxide, while C 60(OH) 24 phototoxicity is mainly due to superoxide.

Autophagy Controls p38 Activation to Promote Cell Survival under Genotoxic Stress

Background: The role of autophagy in genotoxic stress remains poorly understood. Results: Autophagy deficiency sensitizes cells to UVB-induced apoptosis through p62-dependent p38 activation. Conclusion: Inhibition of autophagy promotes apoptosis following UVB damage by increasing p38 activation. Significance: These findings identify the vital role of autophagy in cell survival under genotoxic stress and suggest the function of autophagy in cancer pathogenesis. Deregulated cell survival under carcinogen-induced genotoxic stress is vital for cancer development. One of the cellular processes critical for cell survival under metabolic stress and energy starvation is autophagy, a catabolic process involved in capture and delivery of cytoplasmic components to lysosomes for degradation. However, the role of autophagy following carcinogen-induced genotoxic stress remains unclear. Here we show that UVB radiation, a known human skin carcinogen that operates by causing DNA damage, induced autophagy and autophagic flux through AMP kinase activation. Autophagy deficiency sensitizes cells to UVB-induced apoptosis through increasing p62-dependent activation of the stress-activated protein kinase p38. Compared with normal human skin, autophagy was activated in human squamous cell carcinomas, in association with decreased phosphorylation of p38, and increased phosphorylation of ATR and formation of γ-H2AX, two markers of DNA damage response. Our results demonstrate that autophagy promotes cell survival through suppressing p62-mediated p38 activation and thus may facilitate tumor development under genotoxic stress. These findings suggest that autophagy plays an oncogenic role in epithelial carcinogenesis by promoting cell survival.

PTEN Positively Regulates UVB-Induced DNA Damage Repair

Nonmelanoma skin cancer is the most common cancer in the United States, where DNA-damaging ultraviolet B (UVB) radiation from the sun remains the major environmental risk factor. However, the critical genetic targets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is a predisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cell carcinoma (SCC), levels of PTEN were reduced compared with skin lacking these lesions. Likewise, there was a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCCs, supporting a key role for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity of global genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolonged UVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels in PTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essential genomic gatekeeper in the skin through its ability to positively regulate XPC-dependent GG-NER following DNA damage.

Difference in Phototoxicity of Cyclodextrin Complexed Fullerene [(γ-CyD)2/C60] and Its Aggregated Derivatives toward Human Lens Epithelial Cells

The water-soluble fullerene derivative gamma-cyclodextrin bicapped C(60) [(gamma-CyD)(2)/C(60), CDF0] has several clinical applications, including use as a drug carrier to bypass the blood ocular barriers or a photosensitizer to treat tumors in photodynamic therapy. We have assessed the potential ocular toxicity of (gamma-CyD)(2)/C(60) and its aggregated derivatives induced by UVA and visible light in vitro in human lens epithelial cells (HLE B-3). Cell viability using the MTS assay demonstrated that 2 microM (gamma-CyD)(2)/C(60) was highly phototoxic to HLE B-3 cells with UVA irradiation, while no effect was observed in the presence of visible light or when maintained in the dark. In contrast, the aggregated derivative (CDF150) showed neither cytotoxicity nor any phototoxic effect even at 30 microM with either UVA or visible light irradiation. In lens cells treated with (gamma-CyD)(2)/C(60), phototoxicity was manifested as apoptosis. Singlet oxygen production measurement using the EPR/TEMP trapping technique determined that (gamma-CyD)(2)/C(60) (CDF0) efficiently produced singlet oxygen. The rate of singlet oxygen production decreased with increased aggregation, with no production by the fully aggregated sample formed after 150 min of heating (CDF150). UVA irradiation of HLE B-3 in the presence of (gamma-CyD)(2)/C(60) resulted in a significant rise in intracellular protein-derived peroxides. The singlet oxygen quenchers sodium azide and histidine each significantly protected lens cells against (gamma-CyD)(2)/C(60) photodamage, but lutein and Trolox (vitamin E) did not. Clearly, singlet oxygen is an important intermediate in the phototoxicity of monomeric (gamma-CyD)(2)/fullerene. Our results also demonstrate that UVA-blocking sunglasses can limit the ocular phototoxicity of this nanomaterial, while nontoxic endogenous antioxidants like lutein or Trolox cannot provide adequate protection.