Aminolevulinic acid-photodynamic therapy combined with topically applied vascular disrupting agent vadimezan leads to enhanced antitumor responses

The tumor vascular-disrupting agent (VDA) vadimezan (5,6-dimethylxanthenone-4-acetic acid, DMXAA) has been shown to potentiate the antitumor activity of photodynamic therapy (PDT) using systemically administered photosensitizers. Here, we characterized the response of subcutaneous syngeneic Colon26 murine colon adenocarcinoma tumors to PDT using the locally applied photosensitizer precursor aminolevulinic acid (ALA) in combination with a topical formulation of vadimezan. Diffuse correlation spectroscopy (DCS), a noninvasive method for monitoring blood flow, was utilized to determine tumor vascular response to treatment. In addition, correlative CD31-immunohistochemistry to visualize endothelial damage, ELISA to measure induction of tumor necrosis factor-alpha (TNF-α) and tumor weight measurements were also examined in separate animals. In our previous work, DCS revealed a selective decrease in tumor blood flow over time following topical vadimezan. ALA-PDT treatment also induced a decrease in tumor blood flow. The onset of blood flow reduction was rapid in tumors treated with both ALA-PDT and vadimezan. CD31-immunostaining of tumor sections confirmed vascular damage following topical application of vadimezan. Tumor weight measurements revealed enhanced tumor growth inhibition with combination treatment compared with ALA-PDT or vadimezan treatment alone. In conclusion, vadimezan as a topical agent enhances treatment efficacy when combined with ALA-PDT. This combination could be useful in clinical applications.     

Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea reduces UVB-induced inflammatory responses and infiltration of leukocytes in human skin

Identification of natural products capable of affording protection against UVB radiation-induced inflammatory responses and generation of oxidative stress may have important human health implications. The UVB exposure-induced skin injury and oxidative stress has been associated with a variety of skin disease conditions including photoaging, inflammation and cancer. Tea is a popular beverage consumed worldwide. In several mouse skin models, topical application as well as oral consumption of green tea has been shown to afford protection against chemical and UVB-induced carcinogenesis and inflammatory responses. In the present study, we investigated in human skin, whether topical application of (-)-epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent in green tea, inhibits UVB-induced infiltration of leukocytes (macrophage/neutrophils), a potential source of generation of reactive oxygen species (ROS), and generation of prostaglandin (PG) metabolites. Human subjects were UVB irradiated on sun-protected skin to four times their minimal erythema dosage (MED) and skin biopsies or keratomes were obtained either 24 h or 48 h later. We found that topical application of EGCG (3 mg/2.5 cm2) before UVB (4 MED) exposure to human skin significantly blocked UVB-induced infiltration of leukocytes and reduced myeloperoxidase activity. These infiltrating leukocytes are considered to be the major source of generation of ROS. In the same set of experiments we found that topical application of EGCG before UVB exposure decreased UVB-induced erythema. In additional experiments, we found that microsomes from EGCG pretreated human skin and exposed to UVB, compared to UVB exposure alone, produced significantly reduced PG metabolites, particularly PGE2. The PG metabolites play a critical role in free radical generation and skin tumor promotion in multistage skin carcinogenesis. Careful microscopic examination of skin sections, stained with hematoxylin and eosin, under higher magnification (x400) also revealed that EGCG pretreated and UVB-exposed human skin contained fewer dead cells in the epidermis with comparison to nonpretreated UVB-exposed skin. Taken together, our data demonstrate that EGCG has the potential to block the UVB-induced infiltration of leukocytes and the subsequent generation of ROS in human skin. This may explain the possible mechanism involved in anti-inflammatory effects of green tea. We suggest that EGCG may be useful as a topical agent for protection against UVB-induced ROS-associated inflammatory dermatoses, photoaging and photocarcinogenesis. Further studies are warranted in this direction.     

Nondenatured soy extracts reduce UVB-induced skin damage via multiple mechanisms

UV irradiation results in DNA damage, inflammation and immunosuppression, leading to the development of basal and squamous cell carcinomas. Earlier data show that topical treatment with nondenatured soy extracts reduced the incidence and delayed the development/progression of already-initiated skin tumors in high-risk hairless mice. Here we show that pretreatment with nondenatured soy extracts reduced UVB-induced Thymine-Thymine (TT) dimer formation. In vitro, nondenatured soy extracts enhanced UVB-induced checkpoint kinase-1 (Chk1) activation, suggesting a delay in cell cycle progression that enables longer time for DNA repair. Soy also reduced UVB-induced cyclo-oxygenase-2 (COX-2) expression and prostaglandin E2 secretion, and inhibited p38 MAP kinase activation, suggesting its anti-inflammatory activity. Mice pretreated topically with nondenatured soy extracts had reduced levels of UVB-induced TT dimers and COX-2 expression in their skins compared to UVB alone. The nondenatured soy extracts also inhibited vascular endothelial growth factor-induced endothelial tube formation in Matrigel, suggesting a possible inhibitory effect on angiogenesis and tumor progression. Taken together, nondenatured soy extracts could prevent or reduce UVB-induced skin damage via multiple mechanisms, affecting both the initiation and the progression of skin cancer. These data suggest that topical application of nondenatured soy extracts could potentially reduce the incidence of skin cancer.     

p53 tumor suppressor gene: a critical molecular target for UV induction and prevention of skin cancer

The relationship between exposure to UV radiation and development of skin cancer has been well established. Several studies have shown that UVB induces unique mutations (C-->T and CC-->TT transitions) in the p53 tumor suppressor gene that are not commonly induced by other carcinogens. Our studies have demonstrated that UV-induced mouse skin cancers contain p53 mutations at a high frequency and that these mutations can be detected in UV-irradiated mouse skin well before the appearance of skin tumors. This observation suggested that it might be possible to use p53 mutations as a biologic endpoint for testing the efficacy of sunscreens in photoprotection studies. Indeed, application of SPF 15 sunscreens to mouse skin before each UVB irradiation resulted in reduction in the number of p53 mutations. Because p53 mutations represent an early essential step in photocarcinogenesis, these results imply that inhibition of this event may protect against skin cancer development. This hypothesis was confirmed by our finding that sunscreens used in p53 mutation inhibition experiments also protected mice against UVB-induced skin cancer.     

Photodynamic Therapy with Topical dL-aminolevulinic Acid Delays UV Photocarcinogenesis in Hairless Mice

Abstract— Photodynamic therapy (PDT) with topical application of 8-aminolevulinic acid (ALA) followed by irradiation with visible light (ALA-PDT) is a relatively new and promising experimental treatment of superficial premalignant and malignant skin neoplasms. The purpose of this study was to determine whether ALA-PDT can prevent photocarcinogenesis in hairless mice exposed to solar UV. A total of 140 mice was divided into seven groups of 20 mice each. Group 1: solar-UV exposure. Group 2: solar UV and a cream base + visible light once a week. Group 3: solar UV and ALA-PDT once a week. Group 4: solar UV and ALA-PDT once every second week. Group 5: solar UV and ALA-PDT every fourth week. Group 6: ALA-PDT once a week. Group 7: no treatment. The time to first and to second tumor 1 mm was registered. Predefined endpoints, such as one tumor a 4 mm or an area of small confluent tumors on the back of the mice were criteria for withdrawal from the experiment. The time to first and to second tumor was significantly longer in the ALA-PDT-treated mice than in mice only exposed to solar UV and solar-UV/cream base-visible light (P < 0.005). However, we observed an increased death and accident rate in the ALA-PDT-treated groups compared to the groups not treated with ALA-PDT (chi-square test, P = 0.0250). Significantly more ALA-PDT-treated mice were withdrawn because of a tumor 4 mm ( P = 0.0005). The UV unexposed mice developed no tumors. Repetitive treatments with ALA-PDT delay photoinduced carcinogenesis in mice.     

A Macrocyclic Ruthenium(III) Complex Inhibits Angiogenesis with Down‐Regulation of Vascular Endothelial Growth Factor Receptor‐2 and Suppresses Tumor Growth In Vivo

A macrocyclic ruthenium(III) complex [Ru^III(N₂O₂)Cl₂]Cl ( Ru‐1 ) is reported as an inhibitor of angiogenesis and an anti‐tumor compound. The complex is relatively non‐cytotoxic towards endothelial and cancer cell lines in vitro, but specifically inhibited the processes of angiogenic endothelial cell tube formation and cancer cell invasion. Moreover, compared with known anti‐cancer ruthenium complexes, Ru‐1 is distinct in that it suppressed the expression of vascular endothelial growth factor receptor‐2 (VEGFR2), and the associated downstream signaling that is crucial to tumor angiogenesis. In addition, in vivo studies showed that Ru‐1 inhibited angiogenesis in a zebrafish model and suppressed tumor growth in nude mice bearing cancer xenografts.     

Stage-specific alterations of cyclin expression during UVB-induced murine skin tumor development

We have evaluated the in vivo correlation between the expression of cell cycle markers and skin tumor development in SKH-1 hairless mice in a complete photocarcinogenesis protocol. Irradiated mice developed an average of 16 tumors per animal by week 23 with the average number of carcinomas per mouse being 2.1. The expression of p53 and cyclins A and D1 was confined initially to sporadic single cells and gradually developed into foci of patchy intense staining in the basal and granular layers of UVB-exposed epidermis. p53 was expressed in all the papilloma sections examined, whereas cyclins D1 and A were expressed in 68 and 71% of these lesions, respectively. In UVB-induced squamous cell carcinomas (SCC), p53 was expressed in >90% of the tumors, whereas cyclin D1 was detected in 55% of the lesions, and cyclin A staining was limited to 27%. These immunohistochemical observations were confirmed by Western blotting and protein kinase assays. We observed an early wave of cyclin A overexpression and cyclin A protein kinase activity preceding the appearance of detectable tumors. Cyclin D1 and p53 overexpression were coupled with the development of tumors, and these changes are likely to be relevant to the pathogenesis of these lesions.     

Collagen-EGCG Combination Synergistically Prevents UVB-Induced Skin Photoaging in Nude Mice

Ultraviolet (UV) radiation is a major cause of skin photoaging through generating excessive oxidative stress and inflammation. One of the strategies is to use photo-chemoprotectors, such as natural products with antioxidant and anti-inflammatory properties, to protect the skin from photo damage. The present study investigates the photoprotective potentials of topical administration of unhydrolyzed collagen, epigallocatechin gallate (EGCG), and their combination against ultraviolet B (UVB)-induced photoaging in nude mice. It is found that both the solo and combined pretreatments could recover UVB-induced depletion of antioxidative enzymes, including superoxide dismutase and glutathione peroxidase (GSH-Px), as well as an increase of lipid peroxide malondialdehyde and inflammatory tumor necrosis factor-α. Meanwhile, the UVB-stimulated skin collagen degradation is attenuated significantly with drug treatments, which is evidenced by expression analysis of matrix metalloproteinase-1 and hydroxyproline. Additionally, the mouse skin histology shows that the drug-pretreated groups possess decreased epidermis thickness and normal collagen fiber structure of the dermis layer. These results demonstrate that both EGCG and collagen can protect the skin against UVB-induced skin photoaging. Synergistically, the combination of them shows the maximum prevention to skin damage, showing its potential in the application of anti-photoaging formulation products.     

Novel Aryl‐Modified Benzoylamino‐N‐(5,6‐dimethoxy‐1H‐benzoimidazol‐2‐yl)‐heteroamides as Potent Inhibitors of Vascular Endothelial Growth Factor Receptors 1 and 2

Tumor angiogenesis has become an important target for antitumor therapy, with most current therapies aimed at blocking the vascular endothelial growth factor (VEGF) pathway. The VEGF and its receptors have been implicated as key factors in tumor angiogenesis and are major targets in cancer therapy. A series of aryl‐modified benzoylamino‐N‐(5,6‐dimethoxy‐1H‐benzoimidazol‐2‐yl)‐heteroamides were synthesized from 2‐amino‐5,6‐dimethoxy benzimidazole and aryl‐substituted benzoylamino hetero acids. The new compounds were tested for inhibition of VEGF receptors I and II (VEGFR‐1 and VEGFR‐2). Compound 6e displayed VEGFR‐2 inhibitory activity with a 50% inhibition concentration value as low as 0.020 μM in a homogeneous time‐resolved fluorescence enzymatic assay. VEGFR‐2 active compounds display good activity against VEGFR‐1 as well.     

Noninvasive Assessment of UV-induced Skin Damage: Comparison of Optical Measurements to Histology and MMP Expression

Acute exposure to UV radiation (UVR) causes visible skin damage such as erythema and results in local and systemic immunosuppression while chronic exposure can result in photocarcinogenesis. These deleterious effects can be quantified by histology and by bioassays of key biological markers, including matrix metalloproteinases (MMPs), or tryptophan moieties. We now report our results in quantifying UV skin damage with noninvasive optical methods based on reflectance and fluorescence spectroscopy and compare these noninvasive measurements to histopathology and MMP-13 expression. A solar simulator with spectral output nearly identical to that of solar radiation was developed and used in our experiments. SKH1 hairless mice were exposed to solar-simulated UVR at a total dose of 21 MED delivered over 10 weeks. Changes in oxygenated and deoxygenated hemoglobin were measured by diffuse reflectance spectroscopy, and tryptophan changes were monitored via a fluorescence monitor. Our results show that there is an increase in erythema, skin fluorescence, sunburn cells and MMP-13 after a series of suberythemal doses of UV irradiation on a hairless mouse animal model. Increased skin fluorescence is observed with increasing UV exposure. The levels of MMP-13 increase as the cumulative UV dose increases but their increase does not correspond to noninvasively measured changes.     

Enhanced antitumor chemo-immunotherapy by local co-delivery of chemotherapeutics,immune checkpoint blocking antibody and IDO inhibitor using an injectable polypeptide hydrogel

The efficiency of antitumor immunotherapy is usually limited by the immunosuppressive tumor microenvironment (TME). In this study, we developed a chemo-immunotherapy strategy that is able to improve the immunosuppressive TME for enhancing the antitumor efficacy. The chemo-immunotherapy was achieved by the topical co-delivery of a chemotherapeutic drug, Doxorubicin (DOX), an immune checkpoint blocking antibody targeting programmed cell death protein 1 (aPD-1), and an indoleamine-2,3-dioxygenase (IDO) inhibitor, 1-methyl-d -tryptophan (d -1MT) by using a thermosensitive polypeptide hydrogel. It was revealed that the sustained DOX release from the hydrogel caused the immunogenic cell death (ICD) of B16F10 cells in vitro, and the tumor cell lysates subsequently promoted the activation of dendritic cells (DCs). After intratumoral injection into B16F10 melanoma-bearing mice, the DOX/aPD-1/D-1MT co-loaded hydrogel exhibited enhanced tumor inhibition efficacy and prolonged animal survival time, compared to the DOX/aPD-1/D-1MT mixed solution, DOX-loaded hydrogel or DOX/aPD-1 co-loaded hydrogel. The improvement of immunosuppressive TME and enhancement of antitumor immune response after the local chemo-immunotherapy were demonstrated by the augmented activation of DCs and increased infiltration of CD8⁺ and CD4⁺ T cells, as well as enhanced secretion of pro-inflammatory cytokines. Therefore, the hydrogel-based local chemo-immunotherapy system holds great potential for effective antitumor treatment.     

Dietary Feeding of Opuntia Humifusa Inhibits UVB Radiation‐Induced Carcinogenesis by Reducing Inflammation and Proliferation in Hairless Mouse Model

It has been validated that ultraviolet B (UVB) irradiation induced both squamous and basal cell carcinomas, as a tumor initiator and promoter. Opuntia humifusa is a member of the Cactaceae family which has been demonstrated in our previous study to have a chemopreventive effect in 7, 12‐dimethylbenz[a]anthracene and 12‐O‐tetradecanoylphorbol‐13‐acetate induced skin carcinogenesis models. Therefore, this study was designed to determine the protective effects of O.humifusa against photocarcinogenesis. O. humifusa was administrated to mice as a dietary feeding, following exposure to UVB radiation (180 mJ/cm²) twice a week of 30 weeks for skin tumor development in hairless mice. Dietary O.humifusa inhibited UVB‐induced epidermal hyperplasia, infiltration of leukocytes, level of myeloperoxidase and the levels of proinflammatory cytokines, tumor necrosis factor‐ α (TNF‐α), interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6), in UVB exposed skin. Also, O.humifusa significantly inhibited both protein and mRNA expression level of cyclooxygenase‐2 (COX‐2), nitric oxide synthase (iNOS), proliferating cell nuclear antigen (PCNA) and cyclin D1 compared to the non‐O.humifusa treated group. Collectively, these results suggest that O.humifusa could inhibit photocarcinogenesis in mouse skin and that protective effect is associated with the inhibition of not only UVB‐induced inflammatory responses involving COX‐2, iNOS and proinflammatory cytokines, but also the down‐regulation of UVB‐induced cellular proliferation.     

Involvement of interleukin-10 promoter polymorphisms in nonmelanoma skin cancers-a case study in non-Caucasian skin cancer patients

Interleukin 10 (IL-10) is a potent immunosuppressive cytokine, therefore elevated IL-10 expression has been implicated in inhibition of antitumor immune response. IL-10 gene promoter polymorphism has been shown to be involved in susceptibility to skin cancers, but there has been no report focusing on susceptibility to skin cancers among non-Caucasian populations. We enrolled 129 patients with skin cancers and 50 age- and sex-matched healthy controls between April 2004 and March 2007. Genomic DNA was extracted from patients' blood samples and IL-10 promoter polymorphisms were identified using polymerase chain reaction-restriction fragment length polymorphism or direct sequencing. The distribution of the frequency of allele or haplotype of IL-10 gene promoter in Japanese was quite different from that of Europeans. No significant differences could be demonstrated in the frequency of allele or haplotype of IL-10 gene promoter between the patient group and the control group. However, the frequency of the low-IL-10 expression haplotype was significantly high in Bowen's disease subgroup. The frequency of low expression IL-10 promoter genotype was significantly less (P = 0.009, chi(2) = 6.74) in the group of nonmelanoma skin cancer generated on sun-exposed areas in comparison with that on covered areas. Our results indicated that low expression haplotype of IL-10 in Bowen's disease may inhibit the escape of tumor cells from immune surveillance, resulting in suppression of tumor growth and tumor invasion to the dermis. Moreover, high IL-10-expressing haplotype of IL-10 promoter may be a risk factor for photocarcinogenesis.     

Identification of a Common Pharmacophore for Binding to MMP2 and RGD Integrin: Towards a Multitarget Approach to Inhibit Cancer Angiogenesis and Metastasis

During tumor angiogenesis different growth factors, cytokines and other molecules interact closely with each other to facilitate tumor cell invasion and metastatic diffusion. The most intensively studied as molecular targets in anti-angiogenic therapies are vascular endothelial growth factor (VEGF) and related receptors, integrin receptors and matrix metalloproteinases (MMPs). Considering the poor efficacy of cancer angiogenesis monotherapies, we reasoned combining the inhibition of α_vβ₃ and MMP2 as a multitarget approach to deliver a synergistic blockade of tumor cell migration, invasion and metastasis. Accordingly, we identified a common pharmacophore in the binding cavity of MMP2 and α_vβ₃, demonstrating such approach with the design, synthesis and bioassays of tyrosine-derived peptidomimetics carrying the necessary functional groups to bind to key pharmacophoric elements of MMP2 and α_vβ₃ RGD integrin.     

Combined blockade of HER2 and VEGF exerts greater growth inhibition of HER2-overexpressing gastric cancer xenografts than individual blockade

Gastric cancer overexpressing the human epidermal growth factor 2 (HER2) protein has a poor outcome, although a combination of chemotherapy and the anti-HER2 antibody trastuzumab has been approved for the treatment of advanced gastric cancer. Vascular endothelial growth factor (VEGF) expression in gastric cancer is correlated with recurrence and poor prognosis; however, the anti-VEGF antibody bevacizumab has shown limited efficacy against gastric cancer in clinical trials. In this study, we evaluated the antitumor effects of trastuzumab; VEGF-Trap binding to VEGF-A, VEGF-B and placental growth factor (PlGF); and a combination of trastuzumab and VEGF-Trap in a gastric cancer xenograft model. Although trastuzumab and VEGF-Trap each moderately inhibited tumor growth, the combination of these agents exerted greater inhibition compared with either agent alone. Immunohistochemical analyses indicated that the reduction in tumor growth was associated with decreased proliferation and increased apoptosis of tumor cells and decreased tumor vascular density. The combined treatment resulted in fewer proliferating tumor cells, more apoptotic cells and reduced tumor vascular density compared with treatment with trastuzumab or VEGF-Trap alone, indicating that trastuzumab and VEGF-Trap had additive inhibitory effects on the tumor growth and angiogenesis of the gastric cancer xenografts. These data suggest that trastuzumab in combination with VEGF-Trap may represent an effective approach to treating HER2-overexpressing gastric cancer.     

French Maritime Pine Bark (Pinus maritima Lam.) Extract (Flavangenol®) Prevents Chronic UVB Radiation-induced Skin Damage and Carcinogenesis in Melanin-possessing Hairless Mice

A French maritime pine bark extract, Flavangenol^®, is widely used as a nutritional supplement for protection against atherosclerosis, hypertension, diabetes, etc. Chronic exposure to solar UV radiation damages skin, increasing cutaneous thickness, wrinkling and pigmentation, as well as reducing elasticity, and causes skin cancer. The aim of this study was to examine the effects of flavangenol on skin damage and the incidence of skin tumors caused by long-term UVB irradiation in melanin-possessing hairless mice. The oral administration of flavangenol (60, 200 or 600 mg kg⁻¹, twice daily) significantly inhibited increases in skin thickness, and the formation of wrinkles and melanin granules, as well as increases in the diameter and length of skin blood vessels. Furthermore, it prevented increases in numbers of apoptotic, Ki-67-positive and 8-hydroxy-2′-deoxyguanosine (8-OHdG)-positive cells, and the expression of skin vascular endothelial growth factor (VEGF) induced by chronic UVB irradiation. The effect on these biomarkers was associated with a reduction in the incidence of tumors in mice. The antiphotoaging and anticarcinogenetic activities of flavangenol may be due to inhibition of the expression of Ki-67, 8-OHdG and VEGF through a scavenging effect on reactive oxygen species.     

Identification of a novel class of small-molecule antiangiogenic agents through the screening of combinatorial libraries which function by inhibiting the binding and localization of proteinase MMP2 to integrin alpha(V)beta(3).

The process of new blood vessel growth from existing vasculature, known as angiogenesis, is critical to several pathological conditions, most notably cancer. Both MMP2, which degrades the extracellular matrix (ECM), and integrin alpha(V)beta(3), which contributes to endothelial cell attachment to the ECM, are critically involved in this process. Recent findings have shown that MMP2 is localized in an active form on the surface of invasive endothelial cells based on its ability to directly bind integrin alpha(V)beta(3), suggesting that disrupting this protein--protein interaction may represent a new target for the development of angiogenesis inhibitors. The screening of small molecule libraries led to the identification of compounds which disrupt the MMP2--alpha(V)beta(3) interaction in an in vitro binding assay. A prototypical inhibitor was further found to prevent the degradation of the protein matrix without directly inhibiting MMP2 activity or disrupting the binding of alpha(V)beta(3) to its classical ECM ligand, vitronectin. The synthesis and screening of analogues and substructures of this lead compound allowed the identification of requisite structural features for inhibition of MMP2 binding to alpha(V)beta(3). This led to the synthesis of a more water-soluble derivative which maintains the in vitro biological properties and has potent antiangiogenic and antitumor activity in vivo, validating the target as one useful for therapeutic intervention.