Differential up-regulation of metallothionein isoforms in well-differentiated nasopharyngeal cancer cells in vitro by photoactivated hypericin

Photoactivation of hypericin is known to generate reactive oxygen species and induce phototoxic effects. However, modulation of the cellular antioxidant defense would influence the extent and severity of the photodynamic effects. We have previously shown that hypericin-mediated photodynamic therapy (PDT) induced a significant reduction of Glutathione S-transferase activity. In this study, we investigated the phototoxic effects of hypericin-mediated PDT in nasopharyngeal cancer (NPC) in vitro and analyzed the expression of metallothionein (MT), a family of potential free radical scavengers. HK1 NPC cells were subjected to PDT treatment in vitro, and the effects on cell death were analyzed by flow cytometry (using propidium iodide and Annexin V staining) and transmission electron microscopy. The expression profile of MT-1E and MT-2A isoforms (the only functional MT isoforms expressed in HK1 NPC cells) were determined by quantitative real-time RT-PCR. The results showed that hypericin PDT induced necrotic cell death as evidenced by the absence of a subdiploid peak and decreased Annexin-V fluorescence. Ultrastructural examination verified the presence of cell necrosis. There was a significant up-regulation of MT-1E and MT-2A isoforms six hours following PDT, with an approximately 50-fold rise in the expression level of MT-1E and a 15-fold increase of MT-2A. Hence, despite the up-regulation of MT, cells still succumbed to PDT-induced necrosis. It appears that the oxidative stress induced by PDT overwhelmed the antioxidant defense mechanism such as the alteration of MT levels in tumor cells.     

Hypericin-mediated photodynamic therapy induces lipid peroxidation and necrosis in nasopharyngeal cancer

Photoactivation of hypericin is known to generate singlet oxygen and superoxide anion radicals. Reactive oxygen species (ROS) produced by photodynamic therapy (PDT) has the capacity to induce oxidative damage and tumor destruction. We have previously shown that hypericin-PDT induces tumor shrinkage and regression in the human nasopharyngeal cancer (NPC)/HK1 murine tumor model. In this extended study, we show by electron microscopy that subcutaneously implanted HK1 NPC cells from Balb/c nude mice perished by cell necrosis with hypericin-PDT treatment. There was evidence of cytoplasmic swelling accompanied by loss of cell membrane integrity and autophagic vacuolization of cytoplasm but no nuclear changes. There was also no significant difference in the apoptotic index of control and PDT-treated tumors, when analyzed by in situ end labeling of DNA strand breakage to detect apoptosis. This further supports the observation that cell death in PDT-treated NPC/HK1 tumors was by necrosis. Lipid peroxidative stress analyzed by the malonaldehyde assay was significantly elevated in PDT-treated cells. However, PDT had no effect on the activity of superoxide dismutase, an intracellular antioxidant enzyme. The findings show that hypericin-PDT of nasopharyngeal tumors in vivo induces tumor necrosis with accompanying lipid peroxidation.     

Biodistribution and photodynamic therapy with hypericin in a human NPC murine tumor model

The use of photodynamic therapy (PDT) for the treatment of recurrent and residual nasopharyngeal carcinoma (NPC) has been encouraging. To determine the potential of hypericin as a PDT tool in the treatment of NPC, we investigated the effect of hypericin-mediated PDT on subcutaneously implanted NPC/HK1 tumor cells and the relationship between the biodistribution of hypercin and photodynamic effects. The plasma hypericin level increased rapidly and reached its peak concentration at 1 h after injection. The uptake of hypercin in tumor tissue was maximal 6 h after hypericin administration, at which time the drug concentration in the circulation was low. The efficacy of hypericin-mediated PDT was maximal when light irradiation was performed at 6 h after hypericin administration. Tumor relative regression percentage (RRP) induced by PDT at 1-h interval was comparable to that at 6-h interval, whereas light treatment performed at other time intervals induced less tumor RRP, albeit significant when compared to the control group. Hypericin appears to be an effective photosensitizer for the treatment of NPC. It is likely that hypericin-mediated PDT induces both vascular damage and direct tumor cell killing, thereby bringing about tumor necrosis and shrinkage.     

Silencing of hypoxia-inducible tumor suppressor lysyl oxidase gene by promoter methylation activates carbonic anhydrase IX in nasopharyngeal carcinoma

Lysyl oxidase (LOX) is an oxidative enzyme known to initiate the cross-linking of collagens and elastin, and suggested recently as a tumor suppressor for several tumor types including lung, pancreatic and gastric cancers. Previously we showed that LOX is strongly induced upon hypoxia in nasopharyngeal carcinoma (NPC) cell lines CNE2 and HONE1 but only slightly in HK1 and not in C666-1. Here, we further studied the regulatory mechanism and functions of LOX in NPC. LOX is widely expressed in human normal tissues with variations in expression levels. LOX was expressed in most NPC cell lines except for C666-1, while HK1 and FaDu (laryngeal cancer) only expressed low level of LOX. Methylation analysis showed that the LOX promoter was methylated in C666-1 and partially methylated in HK1. After demethylation with 5-aza-2’-deoxycytidine, LOX expression was reactivated along with increased unmethylated alleles. LOX promoter methylation was detected in 42/49 (85.7%) of NPC primary tumors but only 3/16 (18.75%) of nose swab samples from NPC patients. LOX overexpression reduced the clonogenicity and cell growth of NPC cells, and also inhibited the migration and invasion of the NPC cells. Carbonic anhydrase IX (CA9) mRNA level was obviously decreased in HK1 cells after transfection with LOX. The elevation of CA9 protein upon hypoxia was inhibited in LOX-transfected HK1 cells. The protein levels of an apoptosis marker cPARP were increased in LOX-transfected HK1 cells upon hypoxia treatment. Our data showed that silencing or down-regulation of LOX in NPC was due to its promoter methylation and LOX acts as a tumor suppressor in NPC. LOX silencing would facilitate NPC cells to escape from hypoxia-induced apoptosis and maintains a malignant and metastatic phenotype.     

Photodynamic effects on nasopharyngeal carcinoma (NPC) cells with 5-aminolevulinic acid or its hexyl ester

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Hong Kong and southern China. To explore a new modality of NPC treatment, 5-aminolevulinic acid (ALA) or its hexyl ester (ALA-H) mediated photodynamic therapy (PDT) was studied in vitro. The results show that NPC cells are sensitive to both ALA and ALA-H mediated PDT. However, ALA-H PDT is much more effective at cell inactivation than ALA-PDT, due to a higher efficiency of ALA-H on producing endogenous protoporphyrin (PpIX) in cells. Both apoptosis and necrosis are involved in cell death, but apoptosis plays a major role under the short time incubation of drugs. ALA and ALA-H mediated PDT not only destroy the cells directly, but also inhibit the expression of matrix metalloproteinase-2 (MMP2) in cells, a maker for tumor metastasis. The ALA-H shows promising PDT results on NPC in vitro; therefore it is worth investigating further in vivo for NPC treatment.     

Anti-tumor effect of angiogenesis inhibitor TNP-470 on the human nasopharyngeal carcinoma cell line NPC/HK1.

The efficacy and targeting cells of angiogenesis inhibitor TNP-470 on human squamous cell nasopharyngeal carcinoma (NPC) were investigated. The colorimetric MTT assay was used to evaluate the IC50 values of NPC/HK1 cells and human dermal microvascular endothelial cells (HDMEC) for TNP-470. An NPC human tumor model was built by tumor-bearing nude mice using the NPC cell line of NPC/HK1. TNP-470 (30 mg/kg s.c.) was injected every other day. The results showed that the IC50 of NPC/HK1 cells for TNP-470 was 3.8 times higher than that of HDMEC. A significant difference in tumor volume between control and treatment groups was found after 7 days of treatment and increased thereafter. At the end of the treatment, tumor volume was 773.7 +/- 287.1 mm3 (n = 8) in the control group versus 454.5 +/- 132.8 mm3 (n = 8) in the treatment group (p = 0. 013); the ratio of the mean tumor volume in treated animals to that of control animals was 0.587, resulting a 41.3% decrease in tumor growth. The necrotic area was larger in the treatment group. Physical toxicity did not result from the treatment. These studies suggest that angiogenesis inhibitor TNP-470 is effective in the treatment of squamous cell NPC without obvious toxicity.     

Methylation-associated silencing of miR-9-1 promotes nasopharyngeal carcinoma progression and glycolysis via HK2

Characteristically, cancer cells metabolize glucose through aerobic glycolysis, known as the Warburg effect. Accumulating evidence suggest that during cancer formation, microRNAs (miRNAs) could regulate such metabolic reprogramming. In the present study, miR-9-1 was identified as significantly hypermethylated in nasopharyngeal carcinoma (NPC) cell lines and clinical tissues. Ectopic expression of miR-9-1 inhibited NPC cell growth and glycolytic metabolism, including reduced glycolysis, by reducing lactate production, glucose uptake, cellular glucose-6-phosphate levels, and ATP generation in vitro and tumor proliferation in vivo. HK2 (encoding hexokinase 2) was identified as a direct target of miR-9-1 using luciferase reporter assays and Western blotting. In NPC cells, hypermethylation regulates miR-9-1 expression and inhibits HK2 translation by directly targeting its 3' untranslated region. MiR-9-1 overexpression markedly reduced HK2 protein levels. Restoration of HK2 expression attenuated the inhibitory effect of miR-9-1 on NPC cell proliferation and glycolysis. Fluorescence in situ hybridization results indicated that miR-9-1 expression was an independent prognostic factor in NPC. Our findings revealed the role of the miR-9-1/HK2 axis in the metabolic reprogramming of NPC, providing a potential therapeutic strategy for NPC.     

Molecular effectors and modulators of hypericin-mediated cell death in bladder cancer cells

Photodynamic therapy (PDT) is an anticancer approach utilizing a light-absorbing molecule and visible light irradiation to generate, in the presence of O(2), cytotoxic reactive oxygen species, which cause tumor ablation. Given that the photosensitizer hypericin is under consideration for PDT treatment of bladder cancer we used oligonucleotide microarrays in the T24 bladder cancer cell line to identify differentially expressed genes with therapeutic potential. This study reveals that the expression of several genes involved in various metabolic processes, stress-induced cell death, autophagy, proliferation, inflammation and carcinogenesis is strongly affected by PDT and pinpoints the coordinated induction of a cluster of genes involved in the unfolded protein response pathway after endoplasmic reticulum stress and in antioxidant response. Analysis of PDT-treated cells after p38(MAPK) inhibition or silencing unraveled that the induction of an important subset of differentially expressed genes regulating growth and invasion, as well as adaptive mechanisms against oxidative stress, is governed by this stress-activated kinase. Moreover, p38(MAPK) inhibition blocked autonomous regrowth and migration of cancer cells escaping PDT-induced cell death. This analysis identifies new molecular effectors of the cancer cell response to PDT opening attractive avenues to improve the therapeutic efficacy of hypericin-based PDT of bladder cancer.     

Recombinant human tumor necrosis factor alpha does not potentiate cell killing after photodynamic therapy with a silicon phthalocyanine in A431 human epidermoid carcinoma cells

Photodynamic therapy (PDT) is a novel cancer treatment utilizing a photosensitizer, visible light and oxygen. PDT with the silicon phthalocyanine Pc 4, a new photosensitizer, is highly effective in cancer cell destruction and tumor ablation. The mechanisms underlying cancer cell killing by PDT are not fully understood. Tumor necrosis factor alpha (TNF) is a multifunctional cytokine that has been implicated in photocytotoxicity. We asked whether recombinant human TNF (rhTNF) affects Pc 4-PDT cytotoxicity in A431 human epidermoid carcinoma cells. Co-treatment of A431 cells with various doses of Pc 4-PDT and a sub-lethal rhTNF dose led to a sub-additive reduction in cell survival. In addition, in the presence of Pc 4-PDT or rhTNF, caspase-3 activity and apoptosis were induced. The combined treatment, however, did not potentiate either caspase-3 activity or apoptosis. Similar to previous findings we observed that Pc 4-PDT initiated a time-dependent extracellular TNF accumulation. The data suggest that: a) PDT and rhTNF induce cancer cell killing through different mechanisms; and b) Pc 4-PDT-induced TNF production is a stress response that may not directly affect photocytotoxicity.     

Photodynamic Therapy in Gastroenterology

Introduction

Photodynamic therapy (PDT) is a minimally invasive and promising new modality to combat cancer. Upon illumination and in the presence of oxygen, the tissue-localized nontoxic sensitizer generates cytotoxic reactive oxygen species, and as a result, the selective destruction of a targeted tumor may be achieved. The major advantages of PDT compared to traditional standard of care for cancer treatment are higher selectivity and lower toxicity. The high degree of selectivity offered by this modality has been applied to cancer diagnosis by fluorescence.

Purpose

This article is a review the clinical effectiveness of PDT in the treatment of Barrett’s esophagus and the following cancers: esophageal, gastric, biliary tract, pancreatic, and colon. This review highlights the clinical responses to PDT and discusses the possibility of enhancing the efficacy of treatment by combination with targeted therapy.     

Glucose regulated protein induction and cellular resistance to oxidative stress mediated by porphyrin photosensitization

Photodynamic therapy (PDT) utilizes a tumor localizing porphyrin photosensitizer in the clinical treatment of cancer. At a mechanistic level, porphyrin photosensitization generates reactive oxygen species which initiate oxidative damage to a wide spectrum of biomolecules. Cellular stress proteins are also increased following oxidative stress treatments. In the current study, we examined porphyrin photosensitizing parameters associated with induction of the glucose regulated family of stress proteins. Elevated levels of mRNA encoding glucose regulated proteins (GRPs) as well as increases in GRP protein synthesis were observed for mouse radiation induced fibrosarcoma cells exposed to an extended (16-h) porphyrin incubation prior to light exposure. However, a short (1-h) porphyrin incubation prior to light treatment (designed to produce comparable phototoxicity as PDT using the 16-h porphyrin incubation protocol) was associated with only minimal increases in GRP mRNA levels or GRP protein synthesis. The relationship between GRP levels and PDT sensitivity was examined in radiation induced fibrosarcoma cells pretreated with the calcium ionophore A-23187 in order to overexpress GRPs prior to photosensitization. Resistance to PDT was observed in cells overexpressing GRPs only under photosensitizing conditions associated with the extended porphyrin incubation protocol, and this response was not due to changes in cellular porphyrin uptake. In separate experiments, a transient elevation of GRP mRNA levels was observed in transplanted mouse mammary carcinomas following in vivo PDT treatments. Our results indicate that specific targets of oxidative damage (modulated by porphyrin incubation conditions) instead of generalized cellular exposure to reactive oxygen species are correlated with PDT mediated GRP induction. In this regard, GRP induction may be a useful in vivo biochemical marker of PDT mediated injury. These results also support the hypothesis that GRPs may play a role in modulating sensitivity to cellular stresses including certain types of oxidative injury.     

GST-pi expression correlates with oxidative stress and apoptosis in breast cancer

Glutathione S-transferase (GST) is known to play a key role in the detoxification and reduction of reactive oxygen species (ROS). Thus, we assessed GST activity and GST-pi expression in relation to oxidative stress and apoptosis in breast cancer. Tumor tissues from 32 breast cancer patients were evaluated for GST activity and thiobarbituric acid reactive substances (TBARS) that are by-products of oxidative stress. Four-micron sections of formalin-fixed, paraffin embedded tumors were stained immunohistochemically with anti-GST-pi. Apoptotic cells were detected by in situ end labeling of DNA fragments using a commercial kit. TBARS levels were significantly higher in breast cancers of older patients. GST-pi expression was up-regulated in breast cancers that exhibited higher oxidative stress and associated with higher GST activity. Apoptosis in GST-pi negative tumors was not correlated with GST activity, but GST-pi positive tumors within the same range of oxidative stress showed a reduction in apoptosis as well as an increased GST activity. This correlation was absent in GST-pi positive tumors experiencing higher oxidative stress. GST-pi expression may influence the level of GST activity and delay apoptosis in breast cancer. However, GST-pi expression in tumors with higher levels of oxidative stress may not be sufficient to abrogate the deleterious effects of ROS.     

Functional interaction of Ugene and EBV infection mediates tumorigenic effects

Epstein-Barr virus (EBV) infection is associated with many human neoplasms, in which EBV-derived latent membrane protein-1 (LMP1) appears to be critical, but its exact oncogenic mechanism remains to be defined. To this end, our initial microarray analyses identified a LMP1-inducible gene, Ugene, originally characterized as a binding partner for uracil DNA glycosylase 2, which is highly expressed in malignant colon cancer. In this report, it was found that Ugene, designated herein as LMP1-induced protein (LMPIP), was induced, in a time-dependent manner, in EBV-infected peripheral blood mononuclear cells and LMP1-transfected 293 cells. Functionally, when compared with mock-transfected cells, overexpression of LMPIP in nasopharyngeal carcinoma (NPC) cell lines resulted in a decrease in reactive oxygen species production and maintained mitochondria membrane potential (Δψ) loss induced by H(2)O(2). The NPC cells transfected with LMPIP also showed a decrease in G1 population and an increase in the cell population in sub-G1 and multiploid phase, concomitant with increased levels of cell cycle activators, including cyclin D1 and CDK4. In contrast, silencing of LMPIP expression in the NPC tumor cell lines with short hairpin RNA interference revealed significantly decreased cell population at G1/S phase, while the number of cells in multiploid phase increased. Significantly, NPC cells with LMPIP knock-down also showed a decrease in tumorigenic and transforming activity induced by ectopic LMP1 expression, as determined by analyses of soft agar foci and tumor size in nude mice. Further, elevated LMPIP expression was also noted in cytoplasm and nuclei in EBV-infected NPC tumor cell mass and non-EBV-infected tumor cell lines. These results suggested that LMPIP may have an important mediator role in EBV-mediated neoplasm and may serve as a new target for therapy of tumors induced by EBV infection.     

Role of STAT3/5 and Bcl-2/xL in 2-methoxyestradiol-induced endoreduplication of nasopharyngeal carcinoma cells

2‐methoxyestradiol (2ME2), an endogenous metabolite of 17‐β‐estradiol, has been shown to induce apoptosis and cell cycle arrest in various tumor models. We have previously shown that 2ME2 induced endoreduplication in a well‐differentiated nasopharyngeal carcinoma (NPC) HK‐1 and a poorly differentiated C666‐1 cell line. In the present study, we studied the survival factors involved in 2ME2‐induced endoreduplicating NPC cells. In the HK‐1 cells, knockdown of BcL‐xL expression by siRNA resulted in the reduction of endoreduplication and an increase in the percentage of apoptosis. Further mechanistic study revealed that 2ME2 enhanced the expression of the phosphorylated form of STAT5 (p‐STAT5‐Y694), but not p‐STAT3 (Y705) and p‐STAT3 (S727), in the nucleus of HK‐1 cells. Pre‐treatment of cells with JAK/STAT inhibitor AG490 and STAT5 inhibitor resulted not only in the reduced expression of Bcl‐xL, but also reduced the percentage of endoreduplicating cells. In contrast, 2ME2 enhanced the expression of p‐STAT3 in the poorly differentiated C666‐1 cells. Pharmacological inhibition of STAT3 or Bcl‐2/xL resulted in a decrease in endoreduplication of C666‐1 cells. Taken together, the expression of p‐STAT5 and p‐STAT3 was upregulated in 2ME2‐induced endoreduplicating HK‐1 and C666‐1 cells, respectively. Combination of 2ME2 with Bcl‐2/xL inhibitor is a novel strategy to reduce the formation of endoreduplicating cells during chemotherapeutic treatment of NPC. © 2011 Wiley Periodicals, Inc.     

纳米材料在肿瘤光动力治疗中的应用

光动力治疗(Photodynamic therapy,PDT)是利用光动力学反应,即在分子氧存在的情况下通过特定波长的光照射激活光敏剂,产生包括单线态氧的活性氧物质杀伤肿瘤细胞的一种治疗手段。光动力治疗肿瘤具有创伤小、选择性强、低毒性、适用性好的优点,但是,缺乏理想的光敏剂(Photosensitizer,PS),光源组织穿透深度不足,以及肿瘤组织缺氧等问题已经严重限制了光动力治疗在临床上的应用。纳米材料可解决光动力治疗中遇到的上述问题,因此近来在肿瘤光动力治疗中得以广泛应用,本综述将对纳米颗粒在光动力治疗中的应用进展进行综述。