Can I Use Cbd Oil For Skin Cancer On My Face
J Clin Invest. 2003 Jan 1; 111(1): 43–l.
Inhibition of peel tumor growth and angiogenesis in vivo by activation of cannabinoid receptors
M. Llanos Casanova
oneProject on Cellular and Molecular Biological science and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spaintwo Department of Biochemistry and Molecular Biological science I, Schoolhouse of Biology, Complutense University, Madrid, Espana3 Section of Pathology, Hospital General de Móstoles, Madrid, Spainiv Department of Chemistry, Clemson University, Clemson, South Carolina, United states of america
Cristina Blázquez
1Projection on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Espanaii Department of Biochemistry and Molecular Biology I, Schoolhouse of Biology, Complutense Academy, Madrid, Spain3 Department of Pathology, Hospital General de Móstoles, Madrid, Spainfour Department of Chemistry, Clemson University, Clemson, South Carolina, Us
Jesús Martínez-Palacio
iProject on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spainiii Department of Pathology, Hospital General de Móstoles, Madrid, Spainiv Department of Chemical science, Clemson University, Clemson, South Carolina, Usa
Concepción Villanueva
1Project on Cellular and Molecular Biological science and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spaintwo Section of Biochemistry and Molecular Biological science I, School of Biology, Complutense Academy, Madrid, Spainiii Section of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemical science, Clemson Academy, Clemson, Southward Carolina, United states
M. Jesús Fernández-Aceñero
1Project on Cellular and Molecular Biological science and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense Academy, Madrid, Spainiii Department of Pathology, Hospital General de Móstoles, Madrid, Spainfour Department of Chemistry, Clemson Academy, Clemson, Due south Carolina, United states of america
John W. Huffman
oneProject on Cellular and Molecular Biology and Cistron Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Kingdom of spain2 Department of Biochemistry and Molecular Biological science I, School of Biology, Complutense University, Madrid, Spain3 Section of Pathology, Infirmary General de Móstoles, Madrid, Spain4 Department of Chemistry, Clemson University, Clemson, Southward Carolina, USA
José 50. Jorcano
1Project on Cellular and Molecular Biology and Cistron Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spainiii Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Section of Chemistry, Clemson University, Clemson, Southward Carolina, The states
Manuel Guzmán
oneProject on Cellular and Molecular Biology and Gene Therapy, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Kingdom of spain2 Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spainiii Department of Pathology, Hospital General de Móstoles, Madrid, Spain4 Department of Chemical science, Clemson University, Clemson, South Carolina, Us
Received 2002 Jun 7; Accepted 2002 Nov 19.
Abstract
Nonmelanoma pare cancer is i of the about common malignancies in humans. Different therapeutic strategies for the handling of these tumors are currently being investigated. Given the growth-inhibiting effects of cannabinoids on gliomas and the broad tissue distribution of the two subtypes of cannabinoid receptors (CB1 and CBii), nosotros studied the potential utility of these compounds in anti–skin tumor therapy. Here nosotros show that the CB1 and the CB2 receptor are expressed in normal skin and skin tumors of mice and humans. In cell culture experiments pharmacological activation of cannabinoid receptors induced the apoptotic expiry of tumorigenic epidermal cells, whereas the viability of nontransformed epidermal cells remained unaffected. Local administration of the mixed CBone/CB2 agonist WIN-55,212-2 or the selective CB2 agonist JWH-133 induced a considerable growth inhibition of cancerous tumors generated by inoculation of epidermal tumor cells into nude mice. Cannabinoid-treated tumors showed an increased number of apoptotic cells. This was accompanied by impairment of tumor vascularization, as determined by contradistinct claret vessel morphology and decreased expression of proangiogenic factors (VEGF, placental growth gene, and angiopoietin two). Absconding of EGF-R function was as well observed in cannabinoid-treated tumors. These results support a new therapeutic arroyo for the treatment of peel tumors.
Introduction
The epidermis is a stratified squamous epithelium composed mainly of keratinocytes, whose proliferation and differentiation must be tightly regulated and coordinated. Basal keratinocytes, which are fastened to the basement membrane, are undifferentiated and have proliferative potential. Earlier entering the differentiation plan, they withdraw from the cell cycle and migrate toward the surface of the epidermis, leading to the formation of the outermost layer of the epidermis equanimous of enucleated dead squames, which are continuously shed from the surface of the skin (1). The incidence of both benign and cancerous skin neoplasms has been ascent at an alarming rate for the past several years. Thus, nonmelanoma skin cancer is i of the virtually common malignancies in humans: basal cell carcinomas and squamous jail cell carcinomas represent the vast majority of the cancerous tumors diagnosed (2). These tumors are believed to arise mainly from stem cells of hair follicles (three), and their growth and development seems to rely on an early on outburst of neovascularization (4) in which VEGF (5–7) and EGF-R (8, 9) are essential components. The skin is too a major site for metastasis of internal disease (ii, 10). Early on recognition, biopsy confirmation, and treatment option can reduce patient morbidity. Different types of strategies are currently existence investigated as therapies for the handling of these tumors, including cryotherapy, topical chemotherapeutic agents such as 5-fluorouracil, and photodynamics, the success of which is hampered by limitations such as the poor penetration of molecules into the pare and the difficulty to gain admission to the whole tumor (10–12).
Cannabinoids, the active components of Cannabis sativa linnaeus (marijuana) and their derivatives, exert a wide array of effects on the CNS as well as on peripheral sites such equally the immune, cardiovascular, digestive, reproductive, and ocular systems (thirteen–15). Nowadays, it is widely accepted that most of these effects are mediated by the activation of specific G poly peptide–coupled receptors that are normally bound by a family of endogenous ligands — the endocannabinoids (14, 16, 17). Two different cannabinoid receptors accept been characterized and cloned from mammalian tissues: the "cardinal" CB1 receptor, mostly expressed in encephalon and responsible for cannabinoid psychoactivity (18), and the "peripheral" CBtwo receptor, mostly expressed in the immune system and unrelated to cannabinoid psychoactivity (19). Marijuana and its derivatives have been used in medicine for many centuries, and currently there is a renaissance in the written report of the therapeutic effects of cannabinoids, which constitutes a widely debated issue with aplenty scientific and social relevance. Ongoing research is determining whether cannabinoid ligands may be effective agents in the treatment of, for example, pain and inflammation, neurodegenerative disorders such as multiple sclerosis and Parkinson'southward disease, and the wasting and emesis associated with AIDS and cancer chemotherapy (13–15). In addition, cannabinoids may be potential antitumoral agents owing to their ability to induce the regression of various types of tumors, including lung adenocarcinoma (xx), glioma (21, 22), and thyroid epithelioma (23) in animal models. Although cannabinoids directly induce apoptosis or cell cycle arrest in unlike transformed cells in vitro (24), the involvement of this and other potential mechanisms (e.g., inhibition of tumor angiogenesis) in their antitumoral action in vivo is equally however unknown.
This background prompted u.s.a. to explore whether (a) the skin and skin tumors express cannabinoid receptors; (b) cannabinoid receptor activation exerts a growth-inhibiting action on peel tumors in vivo; and (c) inhibition of angiogenesis is implicated in the anti-tumoral effect of cannabinoids. Our data prove that (a) CB1 and CB2 receptors are present in the skin and pare tumors; (b) local cannabinoid receptor activation induces the regression of peel tumors in vivo; and (c) at to the lowest degree 2 mechanisms may be involved in this activeness: direct apoptosis of tumor cells and inhibition of tumor angiogenesis.
Methods
Cannabinoids.
JWH-133 was prepared in J.Westward. Huffman's laboratory (25). WIN-55,212-two was from Sigma-Aldrich (St. Louis, Missouri, Usa). SR141716 and SR144528 were kindly given past Sanofi-Synthelabo (Montpellier, France).
Jail cell civilization.
The mouse tumorigenic epidermal cell lines PDV.C57 and HaCa4, and the nontransformed epidermal cell lines MCA3D (mouse) and HaCat (homo), were routinely maintained in DMEM supplemented with ten% FCS. Twenty-four hours earlier the experiments, cells were transferred to low serum (0.five%) DMEM. Chief human keratinocytes (Biowhittaker Europe SPRL, Vervier, Belgium) were grown and cultured for the experiments in KGM-2 medium (Biowhittaker Europe SPRL). Stock solutions of cannabinoid ligands were prepared in DMSO. Command incubations had the corresponding DMSO content. No significant influence of DMSO was observed on cell viability at the final concentration used (0.1–0.two%, vol/vol).
Human tumor samples.
Formalin-stock-still, paraffin-embedded, man tumor samples were obtained from the files of the Department of Pathology, Hospital General de Móstoles (Madrid, Spain). Six-micrometer tumor sections were stained with either hematoxylin-eosin or used for immunohistochemistry (run across beneath). The normal pare sample (Figures i and three, skin) came from the face of a 70-year-old man. The slowly growing tumor sample (Effigy 3, BCC) came from the face of a 69-year-quondam woman. The histological analysis revealed the growth of basaloid cell solid nests at the dermoepidermal junction with diagnostic characteristics of basal cell carcinoma. The highly malignant tumor sample (Figures 1 and 3, SCC) came from the retroauricular skin of a 73-year-onetime human being. The histological analysis showed the neoplastic growth of epithelial cells, with squamous differentiation and focal areas of acantholysis.
Western blot analysis of cannabinoid receptor expression in normal skin and skin tumors. (a) CB1 and CBtwo receptor expression in murine and human epidermal prison cell lines, normal peel, and skin tumors. (b) Controls with the anti-CBone or anti-CB2 Ab-blocking peptide are shown (see Methods and Results for explanation). Mouse papillomas were generated by chemical carcinogenesis (every bit described in ref. ix), while mouse squamous cell carcinomas (SCCs) were generated past inoculation of PDV.C57 epidermal tumor cells every bit described in Methods. The source of human samples is described in Methods. Images of representative samples are shown. Similar results were obtained in at to the lowest degree two other blots. Pb, immortalized mouse nontumorigenic cell line derived from SENCAR mice papillomas; HK, human keratinocytes; m, mouse; h, human.
Immunohistochemical analysis of cannabinoid receptor expression in homo normal skin and skin tumors. (a) Immunolocalization of CB1 and CB2 receptors. (b) Controls without primary Ab (only with secondary biotinylated anti-rabbit Ab), as well equally controls with the Ab-blocking peptides, are shown (see Methods and Results for explanation). The source of samples is described in Methods. BCC, basal cell carcinoma; SCC, squamous cell carcinoma; Hf, hair follicle.
Tumor induction in mice.
Cancerous pare tumors were induced in nude (NMRI nu) mice by subcutaneous flank inoculation of 106 PDV.C57 epidermal tumor cells. When tumors had reached an average volume of 500 mm3 (range, 400–600 mm3), a continuous catamenia pump (Alzet 2002; Alza Corporation, Palo Alto, California, Us) was surgically implanted on the flank of every mouse most the site of tumor inoculation. The pump was filled with either vehicle, 1,580 μg of WIN-55,212-2, or i,580 μg of JWH-133 in 236 μl PBS supplemented with five mg/ml BSA and operated at a flux of 0.52 μl/h for 11 days. Tumors were measured with an external caliper and their book calculated every bit (4π/3) × (width/2)2 × (length/2). Cannabinoid treatment did non affect fauna weight gain (data not shown).
Viability and apoptosis in vitro.
Cell viability in the cultures was adamant by the 3-4,five-dimethylthiazol-2,5-diphenyltetrazolium bromide thiazol blue exam. Apoptosis was determined by both TUNEL staining (21) and oligonucleosomal DNA fragmentation (26) according to kit manufacturer's instructions (Boehringer Mannheim GmbH, Mannheim, Frg).
Proliferation and apoptosis in vivo.
For proliferation assays, mice received an intraperitoneal injection of BrdU (120 mg/kg body weight) (Boehringer Mannheim GmbH) two hours before tumor harvesting. Detection of BrdU-positive cells was performed using an anti-BrdU mouse mAb (Boehringer Mannheim GmbH) as described (9). Apoptosis was adamant by TUNEL staining (Boehringer Mannheim GmbH) according to kit manufacturer'south instructions (21).
Western blot analysis.
Particulate cell or tissue fractions were subjected to SDS-Page, and proteins were transferred from the gels onto polyvinylidene fluoride membranes. To determine cannabinoid receptor expression the blots were incubated with polyclonal Ab'south raised in rabbits confronting residues 1–14 of the human/mouse CBane receptor (i:500; kindly given by A. Howlett, N Carolina Central University, Durham, North Carolina, USA) or residues 20–33 of the man/mouse CB2 receptor (1:2,000; Cayman Chemic, Ann Arbor, Michigan, USA) as described (21). Antigen preabsorption experiments were performed by preincubating (37°C, ane 60 minutes) i μl (= 0.five μg) of the anti-CB1 or anti-CB2 Ab and 100 μl PBS with or without 20 μl (= twenty μg) of the corresponding immune peptide (Cayman Chemic). Western blots were subsequently done with the aforementioned Ab dilutions. To determine EGF-R phosphorylation the blots were incubated with anti-phosphotyrosine (1:500; 4G10 mAb; Upstate Biotechnology Inc., Lake Placid, New York, Usa) or anti-keratin 5 (ane:1,000; Berkeley Antibody Co., Richmond, California, U.s.) Ab's, the latter used every bit a loading control. In all cases, samples were subjected to luminography with an enhanced chemiluminescence detection kit (Amersham Life Sciences, Arlington Heights, Illinois, USA). Densitometric analysis of the blots was performed with the Molecular Analyst software parcel (Bio-Rad Laboratories Inc., Hercules, California, United states of america).
Immunohistochemistry.
Tissues were fixed in 10% buffered formalin and embedded in paraffin. Sections of mouse and human skin and tumors were stained with the aforementioned Ab'southward confronting CBi (1:300) and CB2 (1:300) receptors. Control immunostainings using the secondary Ab in the absence of the primary Ab were routinely performed. In addition, antigen preabsorption experiments were carried out with the corresponding blocking peptides as described to a higher place. Immunodetection of blood vessels in cryosections of mouse tumors was performed with an anti-CD31 Ab (ane:40; PharMingen, San Diego, California, United states of america). Sections were incubated with a biotinylated anti-rabbit (CB1 and CBtwo) or anti-rat Ab (CD31) and then with peroxidase-conjugated streptavidin (LSAB Kit Peroxidase; DAKO A/S, Glostrup, Denmark). Ab localization was determined using iii,iii′-diaminobenzidine (Vector Laboratories, Burlingame, California, Usa). Morphometric values were obtained by exam of six 0.11-mmtwo sections per tumor with the image analysis arrangement Leica Qwin (Leica Microsystems Inc., Chantilly, Virginia, USA).
Northern blot assay.
Full RNA was extracted from the tumor samples by the acid guanidinium method (27). The VEGF probe has been described previously (7). The placental growth factor (PIGF) probe was kindly given by Yard. Persico (Istituto Internazionale di Genetica e Biofisica, Naples, Italian republic). Probes for angiopoietin i (Ang1) and angiopoietin two (Ang2) detection were kindly provided past Grand.D. Yancopoulos (Regeneron Pharmaceuticals, Tarrytown, New York, USA). A 1-kb fragment of the v′ region of the hEGF-R cDNA was used as probe for EGF-R detection (9). Ribosomal 7S RNA was used every bit a loading control. Densitometric analysis of the blots was performed with a PhosphorImager using Quantity One software (Bio-Rad Laboratories Inc.).
Statistics.
Results shown represent means ± SD. Statistical analysis was performed past ANOVA with a mail hoc analysis by the Educatee-Neuman-Keuls examination. Data in Table i, Figure 5a, and Figure 6c were analyzed past the Isle of man-Whitney (Wilcoxon) W test to compare medians for nonparametric data.
Cannabinoids inhibit skin tumor growth in vivo. PDV.C57 cells were injected subcutaneously in mice. When tumors had reached the desired size, animals were treated with either vehicle (Co), WIN-55,212-two (WIN), or JWH-133 (JWH), for 11 days. (a) Tumor size (n = 8 for each experimental group). *Significantly unlike (P < 0.01) from control mice. (b) Examples of subcutaneous tumors in the flank of mice after the indicated treatments. (c) Appearance of tumors dissected from mice after the indicated treatments.
Cannabinoids inhibit angiogenesis in skin tumors in vivo. PDV.C57 cells were injected subcutaneously in mice. When tumors had reached the desired size, animals were treated with either vehicle (Co), WIN-55,212-2 (WIN,) or JWH-133 (JWH) for 11 days. (a) Northern blot of the proangiogenic factors VEGF, PIGF, and Ang2. C1, C2; J1, J2; W1, W2 designate tumors from two unlike animals of each experimental grouping, that is, treated with vehicle (Co), JWH-133, or WIN-55,212-2, respectively. OD values relative to those of loading controls are given in capricious units. 7S, Ribosomal 7S RNA. (b) CD31 immunostaining. Note that control carcinomas show dilated blood vessels, while vessels of cannabinoid-treated tumors are narrow. (c) Morphometric analysis of tumor vasculature (n = 4–vi for each experimental group). *Significantly dissimilar (P < 0.05) from control mice.
Table 1
Quantification of apoptotic and proliferative cells in vehicle- and cannabinoid-treated skin carcinomas
Results
Cannabinoid receptors are expressed in skin and skin tumors.
The expression of cannabinoid receptors in epidermal cell lines, normal skin, and peel tumors of mice and humans was examined by Western absorb analysis and immunohistochemistry. Western blot experiments showed that CBane and CB2 receptors were expressed in a number of tumorigenic and nontransformed epidermal prison cell lines of murine and human being origin (Figure 1a). In add-on, the two receptors were present in normal mouse skin equally well every bit in benign (papillomas) and malign (squamous cell carcinomas) mouse peel tumors. Also, CB1 and CB2 receptors were expressed in human being skin, keratinocytes, and carcinomas (Figure onea). To ascertain the specificity of the cannabinoid receptor Ab'due south used in the blotting experiments, antigen preabsorption experiments were carried out with the respective blocking peptides. Every bit shown in Figure 1b, the peptides blocked anti-CB1 and anti-CB2 Ab binding, non only in skin-derived samples but also in other cell types used as well-established controls for the presence of CB1 (rat cortical neurons) (18), CBii (human being promyelocytic HL60 cells) (nineteen), and CB1 plus CB2 (rat C6 glioma cells) (21).
Immunocytochemical analyses showed that in mouse (Figure twoa) and man (Figure 3a) normal skin CB1 and CB2 receptors were mostly nowadays in suprabasal layers of the epidermis and hair follicles. Basal staining was also observed in some desultory regions. CBone and CBii receptor immunoreactivity was likewise evident in both papillomas and squamous cell carcinomas of mouse origin (Figure 2a), as well every bit in human basal cell carcinomas and squamous cell carcinomas (Figure threea). The specificity of the immunolabeling was shown by experiments in which the main Ab was omitted and—as mentioned above for Western blots—by antigen preabsorption experiments carried out with the corresponding blocking peptides (Figures 2b and 3b).
Immunohistochemical analysis of cannabinoid receptor expression in mouse normal skin and skin tumors. (a) Immunolocalization of CB1 and CB2 receptors. (b) Controls without master Ab (only with secondary biotinylated anti-rabbit Ab), equally well every bit controls with the Ab-blocking peptides, are shown (see Methods and Results for explanation). Normal pare came from a 5-twenty-four hour period-erstwhile mouse. Papillomas were generated past chemical carcinogenesis (as described in ref. 9), while SCCs were generated by inoculation of PDV.C57 epidermal tumor cells as described in Methods. Images of representative samples are shown. Similar results were obtained in at to the lowest degree 2 other samples. Hf, pilus follicle; bl, basal layer; sbl, suprabasal layers.
Cannabinoid receptor activation induces skin tumor cell apoptosis.
We tested the functionality of cannabinoid receptors in the consecration of apoptosis in skin tumor cells. The mixed CB1/CBii agonist WIN-55,212-2 decreased the viability of the tumorigenic epidermal cell lines PDV.C57 and HaCa4 (Figure 4a). Values of jail cell viability after WIN-55,212-2 handling (as percentage of the corresponding incubations with no additions) were 74 ± 10 (day 3) and 62 ± vii (day 4) for PDV.C57 cells, and 75 ± 7 (mean solar day 3) and 71 ± 4 (day 4) for HaCa4 cells. Of interest, the cannabinoid was unable to induce any statistically significant modify in the viability of MCA3D and HaCat cells, two nontransformed epidermal cell lines, and of primary human keratinocytes (Figure 4b). The mixed CBane/CBtwo agonist HU-210 and the selective CB2 agonist JWH-133 (K i = 677 nM for CB1 and 3.four nM for CB2; ref, 25) (both at 25 nM) also induced PDV.C57 death to an extent like to that of WIN-55,212-2 (information not shown). WIN-55,212-two–induced decease of PDV.C57 cells occurred by a procedure of apoptosis, every bit adamant by oligonucleosomal DNA fragmentation (Figure fourc) and TUNEL staining (Figure 4d).
Cannabinoid receptor activation induces pare tumor cell apoptosis. (a) The tumorigenic epidermal cell lines PDV.C57 (open symbols) and HaCa4 (closed symbols) were cultured with 25 nM WIN-55,212-ii alone (circles), or in combination with 0.2 μM SR141716 (SR1) (squares), or 0.2 μM SR144528 (SR2) (triangles), and cell viability was determined (n = 5). (b) The nontransformed epidermal cell lines MCA3D (open circles) and HaCat (airtight circles), as well every bit main human being keratinocytes (closed squares), were cultured with 25 nM WIN-55,212-ii, and cell viability was adamant (n = 4). (c and d) PDV.C57 cells were cultured every bit described before, and oligonucleosomal DNA fragmentation (c) (n = 6), and TUNEL staining (d) (i representative experiment of four) were assessed. *Significantly different (P < 0.01) from command incubations.
To evaluate the possible implication of the CB1 and CBii receptors in cannabinoid-induced apoptosis, the effect of selective receptor antagonists was studied. Thus, the CBone adversary SR141716 and the CB2 antagonist SR144528 prevented WIN-55,212-two–induced apoptosis of PDV.C57 cells (Figures iv, a, c, and d), pointing to the involvement of both receptors in the apoptotic action of cannabinoids.
Cannabinoids inhibit skin tumor growth in vivo.
Given the inhibition of tumorigenic epidermal cell survival in culture by cannabinoids, we evaluated the effect of cannabinoid treatment on peel tumor growth in vivo. Tumors generated by inoculation of the highly cancerous PDV.C57 prison cell line were treated with vehicle or WIN-55,212-two. As shown in Effigy 5, a–c, cannabinoid administration blocked the growth of tumor cells in vivo (in ≈75% of the mice treated).
Because cannabinoid-based therapeutic strategies should be as devoid as possible of psychotropic side effects and PDV.C57 express functional CB2 receptors, we administered to mice the selective CBii agonist JWH-133. Previously, we have provided pharmacological, biochemical, and behavioral evidence that JWH-133 activates selectively the CBtwo receptor and does not elicit psychotropic effects in mice (22). Every bit shown in Figure 5, a–c, tumors from JWH-133–treated animals were significantly smaller than those from vehicle-treated controls (in ≈70% of the mice treated).
We adjacent examined whether, as occurs in cultured peel tumor cell lines, cannabinoids induce apoptosis of malignant cells in vivo. As shown in Tabular array 1, quantification of apoptotic cells in tumor sections revealed that treatment with WIN-55,212-2 or JWH-133 increased the number of apoptotic cells. In contrast, the proliferation index did not significantly differ betwixt control and cannabinoid-treated carcinomas.
Cannabinoids inhibit skin tumor angiogenesis in vivo.
Tumors require an acceptable supply of oxygen and nutrients to abound more than a few millimeters. For that purpose they produce proangiogenic factors that promote the germination of new claret vessels (28, 29). We therefore analyzed whether the vascularization of growth-arrested cannabinoid-treated tumors was affected. Northern blot analyses showed that the expression of major proangiogenic factors, namely VEGF, PIGF, and Ang2, was strongly depressed by treatment with WIN-55,212-2 or JWH-133 (Effigy 6a). The mRNA expression of Ang1 and the two antiangiogenic factors, thrombospondin 1 and thrombospondin 2, was not significantly affected by cannabinoid administration (data non shown). Furthermore, although immunostaining of CD31, a marker of endothelial cells, revealed no significant differences in vascular density (number of blood vessels per unit area) between control and WIN-55,212-two– or JWH-133–treated tumors (Figures half-dozen, b and c), important differences were observed when vessel morphology was examined: while control carcinomas showed a network of dilated vessels, cannabinoid-treated tumors displayed a pattern of blood vessels characterized predominantly by narrow capillaries (Figure 6b). Morphometric analyses confirmed that cannabinoid treatment induced a statistically significant decrease in blood vessel size, equally determined by the total area occupied past vessels, the area per vessel, and the vessel larger diameter length (Figure 6c).
Cannabinoids decrease EGF-R activation in skin tumors in vivo.
We have recently establish that in skin carcinomas EGF-R plays an important role in triggering the angiogenic switch necessary for skin tumor growth (9). Thus, we measured the expression levels and activation state of EGF-R in control and cannabinoid-treated skin tumors. While EGF-R mRNA was highly expressed in vehicle-treated tumors, in line with its known overexpression in skin carcinomas (xxx, 31), the levels of EGF-R mRNA in cannabinoid-treated tumors were very depression (Figure 7a). In addition, the degree of EGF-R activation (autophosphorylation) was markedly reduced in cannabinoid-treated tumors (Figure 7b). Moreover, exposure of cultured PDV.C57 cells to WIN-55,212-2 or JWH-133 blunted EGF-R phosphorylation (Figure 7c), supporting the directly touch on of cannabinoids on skin tumor cells.
Cannabinoids inhibit EGF-R activation in skin tumors in vivo. (a and b) PDV.C57 cells were injected subcutaneously in mice. When tumors had reached the desired size, animals were treated with either vehicle (Co), WIN-55,212-2, or JWH-133 for 11 days. Northern (a) and Western blot (b) analyses testify that EGF-R mRNA expression and EGF-R activation (autophosphorylation), respectively, are severely macerated in cannabinoid-treated tumors. One representative experiment of three is shown in each panel. (c) PDV.C57 cells were cultured for 24 hours with either vehicle, 25 nM WIN-55,212-2, or 25 nM JWH-133, and EGF-R phosphorylation was determined by Western blot analysis. One representative experiment of three is shown. OD values relative to those of loading controls are given in arbitrary units. Ribosomal 7S RNA (7S) and keratin five (K5) were used equally loading controls in Northern and Western blots, respectively. PY, phosphotyrosine.
Give-and-take
Here we report that CB1 and CB2 cannabinoid receptors are expressed in normal epidermis and in skin tumors and that both receptors are functional in the induction of apoptosis of skin tumor cells and the regression of peel carcinomas. Information technology is therefore plausible that apoptosis of tumor cells and tumor regression are two causally related events. Even so, our data indicate that cannabinoid antitumoral activity may as well rely on the inhibition of tumor angiogenesis. It has been shown that mouse peel tumor growth and progression depends on critical events leading to epithelial and stromal changes, including the establishment of an active angiogenesis (4). Here, we study that blood vessels developed by cannabinoid-treated carcinomas are small, in line with the finding that claret vessel enlargement constitutes a prominent feature of pare tumor progression (4, 32). Moreover, nosotros evidence that in cannabinoid-treated carcinomas the expression of proangiogenic factors is depressed and that of antiangiogenic factors is unchanged, which fits well with the observations that link skin carcinoma development with a clear imbalance toward positive angiogenic-factor action (six, 7, ix). Ha-ras activation seems to be a critical outcome in mouse peel tumor initiation likewise every bit a major component of the angiogenic response (6) in which VEGF plays a pivotal role (5, nine). Ha-ras activation induces VEGF expression in mouse keratinocytes (six), as well as in other prison cell types (33, 34). Our data also prove that cannabinoid treatment decreases the expression of PIGF (another VEGF family member) and Ang2, and these two proangiogenic factors may act in concert with VEGF because their expression is highly increased since the early stages of tumor evolution (9, 28, 29).
EGF-R participates in the regulation of key epidermal functions (35–38). Moreover, nosotros take shown that in mouse skin carcinomas EGF-R–dependent Ha-ras activation plays a pivotal office in VEGF expression and tumor angiogenesis and growth (ix). Carcinoma growth arising from subcutaneous injection of tumor epidermal cells is a biphasic process. The offset stage of slow growth occurs independently of EGF-R function. Subsequently, an angiogenic switch response mediated by the EGF-R seems to be an essential requirement for complete tumor growth, involving loftier VEGF levels. Other members of the EGF-R family such as HER2 may also exert their relevant anticarcinogenic role via modulation of angiogenesis (39). Here nosotros show that cannabinoid treatment impairs EGF-R role, VEGF expression, and angiogenesis in pare tumors. It is of interest that inhibition of EGF-R function also occurred upon exposure of cultured skin tumor cells to cannabinoids, indicating that the changes observed in EGF-R activity in vivo reverberate a straight bear on of cannabinoids on tumor cells and are not a mere consequence of decreased tumor size. Although at present nosotros cannot plant the machinery for the subtract of EGF-R phosphorylation in cannabinoid-treated tumors, information technology is tempting to speculate that cannabinoid treatment interferes with the tumor angiogenic switch and that this, together with the direct consecration of apoptosis on tumor cells, is a reason for the inhibition of tumor growth in our organization.
Nonmelanoma pare cancer is one of the most mutual malignancies in humans. Different types of strategies are currently being investigated equally therapies for the handling of these tumors, including cryotherapy, topical chemotherapeutic agents such every bit 5-fluorouracil, and photodynamics, the success of which is hampered by limitations such as the poor penetration of molecules into the skin and the difficulty of gaining access to the whole tumor (x–12). The nowadays data point that local cannabinoid assistants may constitute an alternative therapeutic approach for the handling of nonmelanoma skin cancer. Of further therapeutic interest, we testify that skin cells limited functional CB2 receptors. The synergy between CB1 and CB2 receptors in eliciting skin tumor cell apoptosis reported here is withal intriguing because it is non observed in the instance of cannabinoid-induced glioma cell apoptosis (21, 22). In any event, the nowadays study, together with the implication of CB2- or CB2-like receptors in the control of peripheral hurting (40–42) and inflammation (41), opens the attractive possibility of finding cannabinoid-based therapeutic strategies for diseases of the skin and other tissues devoid of nondesired CB1-mediated psychotropic side effects.
Acknowledgments
Nosotros are indebted to Thousand.I. de los Santos for expert technical help and to F. Larcher for discussion and advice. This work was supported by grants from the Ministerio de Ciencia y Tecnología (PM 98-0079 to Chiliad. Guzmán, SAF 98-0047 to J.L. Jorcano, and BMC 2001-1018 to J.L. Jorcano); the Comunidad Autónoma de Madrid (08.1/0079/2000 to Grand. Guzmán); the Fundación Ramón Areces (to M. Guzmán); and the National Institute on Drug Abuse (DA03590 to J.Due west. Huffman).
Footnotes
Disharmonize of interest: The authors have declared that no conflict of interest exists.
Nonstandard abbreviations used: placental growth cistron (PIGF); angiopoietin 1 (Ang1); squamous cell carcinoma (SCC).
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC151833/
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