Wednesday, July 22, 2015

Exploiting Cannabinoid-Induced Cytotoxic Autophagy to Drive Melanoma Cell Death

TL;DR  THC is effective and shows promise in causing melanoma cancer cell death, in addition 1:1 ration of THC:CBD seems to be equally effective.

Jane L Armstrong1,2, David S Hill1, Christopher S McKee1, Sonia Hernandez-Tiedra3, Mar Lorente3, Israel Lopez-Valero3,4, Maria Eleni Anagnostou1, Fiyinfoluwa Babatunde1, Marco Corazzari5, Christopher P F Redfern6, Guillermo Velasco3,4,7 and Penny E Lovat1,7
  1. 1Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle-upon-Tyne, UK
  2. 2Faculty of Applied Sciences, University of Sunderland, Sunderland, UK
  3. 3Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain
  4. 4Instituto de Investigaciones Sanitarias San Carlos (IdISSC), Madrid, Spain
  5. 5Department of Biology, University of Rome “Tor Vergata”, Rome, Italy
  6. 6Northern Institute for Cancer Research, Newcastle University, Newcastle-upon-Tyne, UK
Correspondence: Penny E. Lovat, Dermatological Sciences, Institute of Cellular Medicine, The Medical School, Newcastle University, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK. E mail: penny.lovat@ncl.ac.uk
7These are joint senior authors.
Received 5 November 2014; Revised 9 January 2015; Accepted 21 January 2015
Accepted article preview online 10 February 2015; Advance online publication 12 March 2015
Top

Abstract

Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain <10 class="mb" span="">%
. Novel treatment strategies are therefore urgently required, particularly for patients bearing BRAF/NRAS wild-type tumors. Targeting autophagy is a means to promote cancer cell death in chemotherapy-resistant tumors, and the aim of this study was to test the hypothesis that cannabinoids promote autophagy-dependent apoptosis in melanoma. Treatment with Δ9-Tetrahydrocannabinol (THC) resulted in the activation of autophagy, loss of cell viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.

Abbreviations:

ANOVA, analysis of variance; BDS, botanical drug substance; CBD, cannabidiol; ER, endoplasmic reticulum; siRNA, small interfering RNA; THC, Δ9-Tetrahydrocannabinol
Top

Introduction

Cutaneous melanoma incidence continues to increase, and response rates of patients with metastatic melanoma to current therapy remain poor (Garbe et al., 2011). Identification of driver mutations and development of targeted therapies to BRAF/MEK have revolutionized melanoma therapy, although clinical resistance is inevitable (Chen and Davies, 2014). The emergence of immunotherapies that are able to promote tumor T-cell responses is further changing melanoma management (Wolchok et al., 2010); however, not all patients respond (Prieto et al., 2012). It is therefore clear that there is no consistently beneficial treatment for metastatic melanoma and alternative approaches should be explored.
Autophagy (macroautophagy) is the principal lysosomal-mediated mechanism for the degradation of damaged or long-lived organelles and proteins. Under physiological conditions, autophagy maintains normal turnover of cellular components, as well as responding to metabolic stress, whereas in pathological settings autophagy activation mediates defense against extracellular insults and pathogens (Choi et al., 2013). The current model for the role of autophagy in cancer is that in the early stages of tumor development, quality control by autophagy inhibits tumorigenesis, whereas in advanced cancer autophagy provides energy to meet the increased demands and a means to resist cell death caused by cytotoxic therapy (White, 2012). Preclinical data suggest that lysosomal inhibition can cause tumor regression, and the lysosome inhibitors chloroquine or hydroxychloroquine are now being evaluated in clinical trials either alone or in combination with chemotherapy (Yang et al., 2011). However, recent studies suggest that chloroquine/hydroxychloroquine treatment may promote tumor development (Michaud et al., 2011; Maycotte et al., 2012), questioning the benefit of autophagy inhibition. An alternative approach for autophagy modulation is via exacerbation; although initially this appears counterintuitive to treat advanced cancer, recent evidence suggests that in particular circumstances a consequence of autophagy activation is cell death (Ding et al., 2007; Scarlatti et al., 2008; Tomic et al., 2011; Basit et al., 2013). Therapeutic exploitation of cytotoxic autophagy to drive cancer cell death is therefore an emerging concept for the development of novel cancer treatments.
Cannabinoids are a diverse class of compounds derived from Cannabis sativa, with Δ9-tetrahydrocannabinol (THC) the most relevant owing to its high potency and abundance (Pertwee, 2008). THC exerts its biological effects by mimicking endocannabinoids that bind to and activate two G protein–coupled cannabinoid receptors: CB1 and CB2 (Howlett et al., 2002). CB1/CB2 receptors are expressed in many cancer cell types (Velasco et al., 2012), and cannabinoids are currently being investigated as anticancer agents, including glioblastoma for which THC has shown considerable promise (Velasco et al., 2012). Preclinical data demonstrate that THC exerts its antitumoral action via induction of endoplasmic reticulum (ER) stress, upregulation of the transcriptional coactivator p8 and the pseudo-kinase tribbles homolog 3 (TRIB3), the stimulation of autophagy, and execution of apoptosis (Carracedo et al., 2006; Salazar et al., 2009a, b). Blockade of autophagy prevents THC-induced apoptosis and cell death, indicating that autophagy is upstream of apoptosis and demonstrating the potential of p8/TRIB3-mediated autophagy as a cytotoxic pathway.
Alongside genetic changes, adaption to ER stress and the aberrant control of autophagy have emerged as key drivers of malignancy and therapy resistance in advanced melanoma (Armstrong et al., 2011; Corazzari et al., 2013). The cannabinoid receptors have previously been identified as potential therapeutic targets (Blazquez et al., 2006; Carracedo et al., 2006); hence, targeting ER stress responses combined with cytotoxic autophagy using cannabinoids may represent a valuable therapeutic approach for metastatic melanoma. The aim of this study was to determine whether THC activates cytotoxic autophagy in melanoma cells in vitro and in vivo. Our data suggest a noncanonical mechanism of autophagy-mediated apoptosis, highlighting the potential to harness autophagy for therapeutic benefit in advanced melanoma.
Top

Results

THC activates autophagy and apoptosis in melanoma cells

Characteristic features of early and late stages of autophagy were assessed using LC3 lipidation (LC3-II) and analysis of autophagic flux using chloroquine and visualization of tandem mRFP–GFP (monomeric red fluorescent protein–green fluorescent protein)–tagged LC3 (Kimura et al., 2007), respectively. LC3-II induction was observed in three human melanoma cell lines in response to THC, and LC3-II accumulated further in the presence of chloroquine in both BRAF wild-type (CHL-1) and mutated (A375 and SK-MEL-28) melanoma cells. In addition, increased numbers of LC3-positive autophagosomes (yellow puncta) and autolysosomes (red puncta) and total red fluorescence (CHL-1: t43=4.17, P<0 .001="" a375:="" i="">t
42=3.289, P=0.002) were observed in A375 and CHL-1 cells in response to THC treatment, indicating activation of autophagic flux (Figure 1).
Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorThe Δ9-Tetrahydrocannabinol (THC) induces autophagy in melanoma cells. (ac) A375, SK-MEL-28, and CHL-1 cells were treated with vehicle or THC (4.5 or 5μM) for 24hours in the presence or absence of chloroquine (CQ; 10μM) for the final 2hours, and LC3 and β-actin expression was determined by western blotting. LC3-II expression was quantified and band intensity normalized to β-actin. Data are expressed as fold change relative to the mean LC3-II/β-actin value for a representative experiment and are shown above the western blot (representative data from n=3 independent experiments). (d, e) mRFP–GFP–LC3 expressing (d) A375 or (e) CHL-1 cells were treated with vehicle or THC (4.5μM) for 18hours. Data are representative fluorescent micrographs (bar=20μm) of three independent experiments. (f) Total red fluorescence values were generated from ≥20 cells per treatment condition, from two independent experiments. Pixel intensities were divided by a factor of 106, and data are shown as mean±SD (**P<0 .01="" and="" class="mb" span="">*
**P<0 .001="" cell="" control="" each="" fluorescent="" for="" green="" line="" monomeric="" mrfp="" p="" protein.="" protein="" red="" vs.="">Full figure and legend (165K)Download PowerPoint slide (290 KB)
THC reduced melanoma cell viability in a dose-dependent manner while having little effect on primary melanocytes at doses up to 6μM THC (Figure 2a). Cotreatment of melanoma cells with submaximal concentrations of THC and the pan-caspase inhibitor ZVAD-fmk significantly increased cell viability compared with treatment with THC alone (CHL-1: t22=3.962, P=0.0007; A375: t16=3.74, P=0.0018; Figure 2b), suggesting that cell death is caspase dependent. In addition, the cytochrome c–labeled structures present in vehicle-treated cells were substantially reduced in THC-treated cells (Figure 2c), indicating apoptosis activation.
Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorThe Δ9-Tetrahydrocannabinol (THC) induces apoptosis of melanoma cells. (a) Primary melanocytes, A375, and CHL-1 cells were treated with vehicle or THC (3–10μM) for 24hours. (b) A375 (i) or CHL-1 (ii) cells were treated with vehicle or THC (5μM) in the presence or absence of ZVAD (20μM) for 24hours. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data generated in triplicate were expressed relative to the mean of vehicle-treated cells in each experiment, for three independent experiments, and shown as mean±SD (t-test; **P<0 .01="" and="" class="mb" span="">*
**P<0 .001="" class="mb" span="" thc="" vs.="">+ZVAD). (c) A375 cells were treated with THC (5μM) for 24hours. Data are representative fluorescent micrographs of cytochrome c immunostaining (bar=20μm) of three independent experiments.Full figure and legend (143K)Download PowerPoint slide (266 KB)

THC-induced apoptosis is dependent on autophagy

In glioma, THC activates apoptosis via a mechanism involving TRIB3 and Beclin-1-dependent autophagy (Carracedo et al., 2006; Salazar et al., 2009b). The small interfering RNA (siRNA)-mediated knockdown of TRIB3 significantly prevented loss of cell viability in response to THC in A375 cells (one-way analysis of variance (ANOVA); F5, 48=5.053, P=0.001; THC compared with vehicle treatment in siCtrl cells; Tukey’s P<0 .05="" a="" href="http://www.nature.com/jid/journal/v135/n6/suppinfo/jid201545s1.html">Supplementary Figure S1
online), demonstrating that TRIB3 mediates THC-induced cell death. Furthermore, siRNA-mediated knockdown of Atg7 prevented THC-induced accumulation of LC3-II in the presence of chloroquine (one-way ANOVA; F11, 24=6.878, P<0 .001="" chloroquine="" class="mb" compared="" span="" with="">+THC-treated siCtrl cells contrast P<0 .05="" a="" href="http://www.nature.com/jid/journal/v135/n6/full/jid201545a.html#fig3">Figure 3a and b). THC-induced caspase 3 cleavage was also inhibited by Atg7 knockdown in A375 (Figure 3a), as well as in CHL-1 and SK-MEL-28 cells (CHL-1: one-way ANOVA; F5, 12=6.57, P=0.004; THC compared with vehicle-treated siCtrl cells; Tukey’s P<0 .05="" anova="" f="" one-way="" sk-mel-28:="" sub="">3, 8=4.646, P=0.037; THC compared with vehicle-treated siCtrl cells; Tukey’s P<0 .05="" a="" href="http://www.nature.com/jid/journal/v135/n6/suppinfo/jid201545s1.html">Supplementary Figure S2 online). In addition, THC treatment resulted in a significant loss of melanoma cell viability only in the absence of chloroquine or Atg7 siRNA (Figure 3 and Supplementary Figure S2 online; post hoc tests, Games–Howell, or Tukey’s P<0 .01="" a375="" a="" alone="" and="" atg7="" cell="" cells="" chl-1="" downregulation="" effect="" had="" however="" i="" in="" knockdown="" loss="" no="" of="" on="" resulted="" s="" significant="" sk-mel-28="" ukey="" viability="">P<0 .001="" a="" href="http://www.nature.com/jid/journal/v135/n6/suppinfo/jid201545s1.html">Supplementary Figure S2a online), suggesting that basal autophagy is required to maintain viability in these cells. Collectively, these data demonstrate that THC-induced apoptosis of melanoma cells requires TRIB3 and is mediated by Atg7-dependent autophagy.
Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorThe Δ9-Tetrahydrocannabinol (THC)-induced apoptosis requires autophagy. (ac) A375 cells were transfected with small interfering RNAs (siRNAs) for Atg7 (siAtg7) or with a nonsilencing control siRNA (siCtrl) before treatment with vehicle or THC (4.5, 5μM) for 24hours in the presence or absence of chloroquine (CQ; 10μM) for the (a, b) final 2hours or for (c) 24hours. (a, b) Atg7, LC3, cleaved caspase 3, and β-actin expression were determined by western blotting. LC3-II expression was quantified and band intensity normalized to β-actin. Data are expressed as fold change relative to the mean LC3-II/β-actin value for each experiment, for three separate experiments (mean±SD, n=3). (c) Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data generated in triplicate were expressed relative to the mean of vehicle-treated siCtrl cells in each experiment, for three independent experiments, and shown as mean±SD (*P<0 .05="" class="mb" span="">*
*P<0 .01="" and="" i="">***P<0 .001="" cells="" p="" sictrl="" vehicle-treated="" vs.="">Full figure and legend (121K)Download PowerPoint slide (248 KB)
Beclin-1 promotes autophagy induction and autophagosome formation (Russell et al., 2013); however, Beclin-1-independent autophagy has been reported (Scarlatti et al., 2008; Grishchuk et al., 2011). Unlike in glioma cells, Beclin-1 knockdown did not prevent THC-induced LC3-II accumulation or caspase 3 cleavage (t4=4.494, P=0.011; Supplementary Figure S3a online) in A375 cells (Figure 4a), and THC treatment resulted in a significant loss of cell viability under both control and Beclin-1 knockdown conditions (Welch ANOVA; F5, 19.63=94.53, P<0 .001="" cells="" compared="" games="" i="" owell="" shrna="" thc="" treatments="" vehicle-treated="" with="">P
<0 .001="" a="" href="http://www.nature.com/jid/journal/v135/n6/full/jid201545a.html#fig4">Figure 4a i) and ii)). These results suggest that autophagy and subsequent apoptosis occur independently of Beclin-1. Furthermore, knockdown of the Beclin-1-interacting protein Ambra1 failed to prevent THC-induced LC3-II induction and caspase 3 cleavage (t4=6.097, P=0.004; Supplementary Figure S3b online and Figure 4b). The effect of Ambra1 knockdown alone on A375 viability was variable; however, THC treatment resulted in a significant loss of cell viability under both control (t16=9.44, P<0 .001="" ambra1="" and="" conditions="" i="" knockdown="">t16=10.61, P<0 .001="" a="" href="http://www.nature.com/jid/journal/v135/n6/full/jid201545a.html#fig4">Figure 4biii). Collectively, these data suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells that is dependent on Atg7 but not Beclin-1 or Ambra1.
Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorThe Δ9-Tetrahydrocannabinol (THC)-induced autophagy and cell death is not dependent on Beclin-1 or Ambra1. (a) A375 cells stably expressing short hairpin RNA (shRNA) for Beclin-1 (shBeclin1) or a nonsilencing control shRNA (shCtrl) were treated with vehicle or THC (4.5 and 5μM) for 48hours in the presence or absence of chloroquine (CQ; 10μM) for the final 2hours (i). Beclin-1, LC3, cleaved caspase 3, and β-actin expression were determined by western blotting (i). Cell viability (ii) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data generated in triplicate were expressed relative to the mean of vehicle-treated shCtrl cells in each experiment, for three independent experiments, and shown as mean±SD (***P<0 .001="" b="" cells="" shctrl="" vehicle-treated="" vs.="">b
) A375 cells stably expressing shRNA for Ambra1 (shAmbra1) or a nonsilencing control shRNA (shCtrl) were treated with vehicle or THC (5μM) for 24hours in the presence or absence of CQ (10μM) for the final 2hours (i). Ambra1, LC3, cleaved caspase 3, and β-actin expression were determined by western blotting (i). Cell viability (ii) was determined by the MTT assay. Data generated in triplicate were normalized to the mean of vehicle-treated cells for each shRNA in each experiment, for three independent experiments, and shown as mean±SD (***P<0 .001="" cells="" each="" for="" p="" shrna="" vehicle-treated="" vs.="">Full figure and legend (109K)Download PowerPoint slide (253 KB)

Cannabinoid treatment stimulates autophagy and apoptosis and the abrogation of melanoma growth in vivo

A 1:1 mixture of submaximal doses of THC–botanical drug substance (BDS) and the nonpsychoactive cannabinoid cannabidiol (CBD) –BDS, a laboratory mimic of the clinical cannabinoid Sativex (Sat-L) (an oromucosal spray of standardized cannabis extract comprising equal amounts of THC and CBD (GW Pharmaceuticals)), reduced glioma growth in vivo to the same extent as an identical dose of THC (Torres et al., 2011). Treatment of melanoma cells with THC+CBD resulted in a substantial loss of melanoma cell viability at a concentration of 1μM THC+1μM CBD in CHL-1, A375, and SK-MEL-28 cells compared with equivalent concentrations of THC, whereas temozolomide had little effect (Figure 5). Temozolomide is an alkylating agent currently indicated as standard single-agent chemotherapy for metastatic melanoma. The in vivo relevance of these findings was evaluated in the context of BRAF wild-type melanoma tumors, for which there is a particular demand for novel therapeutic approach in the absence of targeted therapies in this tumor group. CHL-1 xenograft tumors were treated for 20 days with temozolomide, THC, or Sativex-L. Both THC and Sativex-L significantly inhibited the growth of xenografts (one-way ANOVA; F3, 16=9.347, P=0.001; THC or Sat-L compared with vehicle, Sat-L compared with Temozolomide: P<0 .05="" a="" href="http://www.nature.com/jid/journal/v135/n6/full/jid201545a.html#fig6">Figure 6a
). Tumors removed from animals at the time of killing were processed for immunohistochemical analysis of proliferative activity (Ki67), apoptosis (TUNEL), and autophagy (LC3). Ki67 fluorescence differed significantly between drug treatments (Welch ANOVA; F3, 16.28=61.363, P<0 .001="" a="" href="http://www.nature.com/jid/journal/v135/n6/full/jid201545a.html#fig6">Figure 6b) and was significantly reduced in tumors from animals treated with temozolomide, THC, or Sativex-L compared with control animals (Games–Howell; P≤0.001), as well as in tumors from animals treated with THC or Sativex-L compared with those treated with temozolomide (Games–Howell; P<0 .001="" also="" anova="" between="" differed="" f="" fluorescence="" one-way="" significantly="" sub="" treatments="" tunel="">3, 32=13.31, P<0 .001="" and="" animals="" control="" from="" higher="" i="" in="" or="" s="" sativex-l="" than="" thc="" treated="" tumors="" ukey="" was="" with="">P≤0.001) and higher in tumors from animals treated with Sativex-L compared with those treated with temozolomide (Tukey’s P<0 .05="" a="" href="http://www.nature.com/jid/journal/v135/n6/full/jid201545a.html#fig6">Figure 6c). Correspondingly, LC3 fluorescence differed between treatments (one-way ANOVA; F3, 32=3.539, P<0 .05="" animals="" compared="" from="" i="" in="" increased="" lc3="" or="" s="" sativex-l="" significantly="" staining="" temozolomide="" treated="" tumors="" ukey="" vehicle="" with="">P≤0.05, Figure 6d). Staining for Ki67, TUNEL, and LC3 was not significantly different in tumors from animals treated with THC compared with Sativex-L. Collectively, these data suggest that THC and Sativex-L are more effective than temozolomide in terms of apoptosis induction and antitumor response, further validating the therapeutic relevance of cannabinoid treatment for melanoma.
Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorCannabinoids inhibit melanoma cell viability in vitro. (ac) CHL-1 (a), A375 (b), or SK-MEL-28 (c) cells were treated with temozolomide (Temo; 10–50μM), Δ9-Tetrahydrocannabinol (THC; 1–5μM), or THC+CBD (0.5μM THC+0.5μM CBD to 2.5μM THC+2.5μM CBD) for 48hours. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data generated in triplicate were expressed relative to the mean of vehicle-treated cells for each drug treatment in each experiment, for three independent experiments, and shown as mean±SD for a representative experiment. CBD, cannabidiol.
Full figure and legend (109K)Download PowerPoint slide (227 KB)

Figure 6.
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the authorThe Δ9-Tetrahydrocannabinol (THC) and Sativex-like cannabinoids promote autophagy and antitumor responses in melanoma xenografts. Athymic nude mice were injected subcutaneously in the right flank with CHL-1 melanoma cells. When tumors reached a 250mm3 size, mice were treated daily for 20 days with vehicle, temozolomide (TMZ; 5mgkg−1; local administration), THC (15mgkg−1; oral administration), or Sativex-like (Sat-L; 7.5mgkg−1 THC–BDS+7.5mgkg−1 CBD–BDS; oral administration). (a) Tumor volumes were measured daily. Each point is the mean from ≥5 tumors±SD and is expressed relative to the tumor volume on day 1 of treatment. (bd) Immunohistochemical analysis of CHL-1 xenograft tumors treated with temozolomide, THC, or Sat-L. (b–d) Micrographs of tumor sections stained for (b) Ki67, (c) TUNEL, or (d) LC3. In each, green is staining for Ki67, TUNEL, or LC3, and red is the TO-PRO-3 counterstain. The bar graphs below each set of micrographs summarize the data analysis from tumor sections (bar=20μm). Total fluorescence values (total fluorescence (LC3/Ki67/TUNEL)/total nuclear fluorescence) were generated in triplicate for 3 tumors in each treatment group. Data are expressed as fold change relative to the mean value obtained from control animals, from two independent staining analyses, and shown as mean±SD (**P<0 .01="" and="" class="mb" span="">*
**P<0 .001="" animals="" control="" p="" vs.="">Full figure and legend (302K)Download PowerPoint slide (428 KB)
Top

Discussion

It is apparent that autophagy modulation may offer considerable benefit in cancer treatment; however, potential drawbacks to autophagy inhibition have recently been identified (Michaud et al., 2011; Maycotte et al., 2012; Takahashi et al., 2012; Rosenfeldt et al., 2013). Emerging evidence indicates that activation of autophagy can, in some circumstances, promote cell death; stimulation of cytotoxic autophagy therefore represents an alternative approach to autophagy modulation (Ding et al., 2007; Scarlatti et al., 2008; Salazar et al., 2009b; Grishchuk et al., 2011; Tomic et al., 2011; Basit et al., 2013). Here, we show that the cannabinoid THC exerts its antitumor effect on melanoma cells via activation of noncanonical autophagy and subsequent apoptosis, suggesting that cannabinoids may be of clinical benefit for metastatic melanoma.
The molecular mechanisms connecting autophagy to cell death remain poorly understood (Shen and Codogno, 2011); however, reports describing autophagy-dependent apoptosis (Ding et al., 2007; Salazar et al., 2009b; Grishchuk et al., 2011; Tomic et al., 2011) suggest multiple interactions between autophagic and apoptotic machinery. THC exerts its effect via the de novo synthesis of the sphingolipid ceramide, leading to the activation of ER stress, TRIB3-dependent inhibition of Akt/mTORC1 signaling, and autophagy-mediated apoptosis (Carracedo et al., 2006; Salazar et al., 2009b). TRIB3 has been identified as a key switch between cell survival and apoptosis during stress responses (Shimizu et al., 2012), and the participation of TRIB3 in the melanoma response to THC may direct cellular fate toward apoptosis in the context of ER stress–induced autophagy (Salazar et al., 2013).
Autophagy inhibition using both molecular and pharmacological approaches prevented THC-induced autophagy and apoptosis of melanoma cells. However, THC-induced autophagy was not prevented by knockdown of Beclin-1, suggesting that, in contrast to glioma, noncanonical autophagy mediates apoptosis in response to THC in melanoma. Beclin-1-independent autophagy may promote caspase-independent cell death (Scarlatti et al., 2008; Basit et al., 2013) as well as apoptosis (Grishchuk et al., 2011; Tomic et al., 2011), suggesting that autophagy mechanisms not involving Beclin-1 exist that interact with cell death machinery. Interestingly, in contrast to previous studies (Salazar et al., 2009b), THC-induced autophagy was also independent of Ambra1 in melanoma cells. Ambra1 is a Beclin-1 interacting protein that promotes autophagy by stabilizing Beclin-1 complexes (Fimia et al., 2007); although supporting the concept of Beclin-1-independent autophagy activation in response to THC, these data highlight the complex regulation of autophagy that likely occurs in a cell type- and context-specific manner.
Tumor-selective killing can be achieved by targeting pathways that are differentially regulated in cancer cells compared with normal cells. In this respect, we have shown that normal human melanocytes are resistant to THC at concentrations that cause cell death in melanoma cells. This is consistent with studies showing that cancerous cells are more sensitive to THC and other cannabinoid receptor ligands compared with their nontransformed counterparts, despite the presence of functional CB receptors (Velasco et al., 2012). Together with previous studies demonstrating an effect of synthetic ligands of cannabinoid receptors in melanoma (Blazquez et al., 2006), these data support clinical evaluation of cannabinoids in advanced-stage disease. Furthermore, THC activates autophagy and apoptosis in both BRAF wild-type and mutated melanoma cell lines, suggesting that despite autophagy deregulation (Armstrong et al., 2011; Corazzari et al., 2013), THC is likely effective in melanoma tumors regardless of BRAF mutation status. Furthermore, our findings show that THC is able to reduce melanoma cell viability and tumor xenograft growth alone, but when lower doses of THC are combined with CBD the antitumor effect was enhanced in vitro and was at least equally effective as the higher dose of single-agent THC in vivo. Moreover, CBD induces apoptosis via the production of reactive oxygen species and caspase activation in cancer cells (Massi et al., 2006; Shrivastava et al., 2011), indicating that THC and CBD engage different molecular machineries that cooperate to promote tumor cell death (Shrivastava et al., 2011; Torres et al., 2011).
In summary, these data highlight the potential for cannabinoid-induced cytotoxic autophagy as an effective strategy to drive melanoma cell death, supporting the clinical evaluation of Sativex for the treatment of metastatic disease.
Top

Materials and Methods

Cell culture, viability assays, and drug treatment

Melanoma cell lines CHL-1, A375, and SK-MEL-28 were obtained from the ATCC (Manassas, VA) in 2006 and cultured as described previously (Armstrong et al., 2007). Cell lines were verified by melan A staining (Flockhart et al., 2009) with B-RAF/NRAS mutational status confirmed using Custom TaqMan SNP genotyping assays (Hiscutt et al., 2010) (Applera Europe BV, Life Technologies, Paisley, UK) (last tested February 2014). Before drug treatment, culture medium was changed to 0.5% fetal bovine serum medium. Temozolomide (OSI Pharmaceuticals, Melville, NY) and ZVAD-fmk (benzyloxycarbonyl-V-A-D(OMe)-fluoromethylketone) (Tocris Bioscience, Bristol, UK) were added in DMSO, and chloroquine (Sigma-Aldrich Ltd, Poole, UK) was added in water. For in vitro experiments, pure THC (THC Pharm, Frankfurt, Germany) and CBD (synthesized by Professor Raphael Mechoulam (Hebrew University of Jerusalem) and kindly provided by Dr Javier Fernandez Ruiz (Complutense University, Madrid, Spain)) were prepared in DMSO. Control incubations contained the same amount of DMSO (0.1–0.2% v/v). For treatment with THC+CBD, pure THC and pure CBD were mixed 1:1 (w/w). Analysis of cell viability was performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma). For in vivo experiments, THC–BDS (THC content 67.6% w/w; CBD content 0.3% w/w; other individual plant cannabinoids <1 .5="" class="mb" span="">%
w/w) and CBD–BDS (CBD content 65.4% w/w; THC content 2.5% w/w; other individual plant cannabinoids <1 .7="" class="mb" span="">% w/w) were provided by GW Pharmaceuticals (Cambridge, UK). THC–BDS and CBD–BDS were provided as a resin, dissolved in ethanol (100mgml−1), dried, and prepared in DMSO. A 1:1 (w/w) preparation of THC–BDS and CBD–BDS was used to mimic Sativex (Sat-L). For oral administration, THC or Sat-L was solved in 100μl of sesame oil.

Western blotting and reverse transcription–PCR analysis

Preparation of whole-cell lysates and western blotting for LC3B, cleaved caspase 3 (Cell Signaling Technology, Leiden, The Netherlands), Atg7 (Santa Cruz Biotechnology, Heidelberg, Germany), Beclin-1 (BD Biosciences, Oxford, UK), and Ambra1 (Novus Biologicals, Cambridge, UK) all diluted 1:4,000, and β-actin (Sigma) diluted 1:30,000, were performed as described previously (Armstrong et al., 2007). Total RNA was isolated from cells using the RNeasy Mini Kit with DNase digestion (Qiagen, Manchester, UK) according to the manufacturer’s protocol. Reverse transcription–PCR was performed using the Access Reverse transcription–PCR system (Promega, Southampton, UK) using primers for TRIB3 (Carracedo et al., 2006) or β-actin (Armstrong et al., 2005). PCR products were analyzed by electrophoresis on ethidium bromide–stained 2% agarose gels and DNA visualized by exposure to UV light.

siRNA transfections

The siRNA for Atg7 (HSS116182), TRIB3 (sc-44426, Santa Cruz Biotechnology), or a nontargeting siRNA (Negative control Low GC Stealth RNAi) (Stealth RNAi, Life Technologies, Paisley, UK) were transfected into cells in OPTIMEM containing siRNA (2–4nM) using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s specification for reverse transfection. After 24hours, the medium was replaced with DMEM containing 0.5% fetal bovine serum and cells treated with drugs as appropriate.

Retroviral or lentiviral infection

Retroviral expression of mRFP–GFP–LC3 (provided by T Yoshimori, Osaka University, Osaka, Japan) (Kabeya et al., 2000; Kimura et al., 2007) was performed as described previously (Armstrong et al., 2011). Lentiviral expression of shAmbra1 or a nontargeting sequence shCtrl (MISSION shRNA, Sigma) was performed by cotransfection of 7.5μg lentivirus vector (pLKO.1-puro) with 2.5μg of an expression plasmid for the vesicular stomatitis virus G protein into 293 cells using the calcium precipitation method. After 48hours, melanoma cells were incubated with virus-containing supernatant supplemented with polybrene (4μgml−1) for 6–8hours and selected for puromycin resistance. Lentiviral expression of shBeclin-1 (V3LHS_349509, Dharmacon, Chalfont St Giles, UK) or a nontargeting sequence shCtrl (RHS4346, Dharmacon) was performed by cotransfection of 20μg lentivirus vector with 5μg of an expression plasmid for the viral envelope (pMD2.G) and 15μg packaging plasmid (pCMVdelta8.91) into HEK293T cells using the calcium precipitation method. After 72hours, melanoma cells were spin transduced (1.5hours, 1,200r.p.m., Harrier 15/80, DJB Labcare, Newport Pagnell, UK) with virus-containing supernatant supplemented with polybrene (4μgml−1) and selected for puromycin resistance.

Confocal microscopy

Cells were grown on glass coverslips before fixation in 4% paraformaldehyde. For immunolabeling, cells were incubated with 0.2% Triton X-100 before incubation with anti-cytochrome c (BD Biosciences) at room temperature for 1hour (McGill et al., 2005). Secondary labeling was performed with Oregon Green 488 conjugated to anti-rabbit IgG (Life Technologies). Nuclei were counterstained with TO-PRO-3 iodide (Life Technologies). Cells were imaged under a Leica TCS SP II laser-scanning confocal microscope with LCS Lite 2.61 software (Leica Microsystems, Milton Keynes, UK), using a 63 × oil objective.

Xenograft mouse model and immunohistochemical analysis

Athymic nude (nu/nu) 5-week-old male mice (Harlan Iberica Laboratory, Madrid, Spain) were inoculated by subcutaneous injection of 7.5 × 106 CHL-1 cells in 100μl phosphate-buffered saline containing 0.1% glucose. On establishment of tumors 250mm3 in volume, mice were randomized into four treatment groups (5–8 mice per group) and treated by daily administration for 20 days with temozolomide (5mgkg−1, local peritumoral injection), THC (15mgkg−1, oral gavage), or Sativex (7.5mgkg−1 THC–BDS+7.5mgkg−1 CBD–BDS, oral gavage). The control group was treated with 100μl of vehicle (sesame oil). Caliper measurements of tumor length (l) and width (w) were taken each day, and tumor volume was calculated as (4π/3) × (w/2)2 × (l/2). Mice were humanely killed on the final day of treatment, and tumors extracted and snap frozen in liquid nitrogen before storage at −80°C. Frozen sections (6μm) prepared on (3-Aminopropyl)triethoxysilane (Sigma)-coated glass slides were fixed in 4% paraformaldehyde before staining by TUNEL or with a Ki67 antibody (ab-15580, Abcam, Cambridge, UK) as previously described (Hill et al., 2009). For LC3 immunolabeling, frozen sections were fixed in acetone and incubated with anti-LC3B (ab48394, Abcam) for 1hour at room temperature. Secondary labeling was performed with Oregon Green 488 conjugated to anti-rabbit IgG (Life Technologies). Cells were imaged under a Leica TCS SP II laser-scanning confocal microscope with LCS Lite 2.61 software (Leica Microsystems), using a 63 × oil objective. All procedures involving animals were performed according to Spanish and European regulations and were approved by the ethical committee for animal experimentation from Complutense University.

Statistical analysis

Images of LC3, Ki67, and TUNEL staining were analyzed using Velocity (v4.3.1) (Improvision, Perkin Elmer, Coventry, UK). Total fluorescence was obtained by multiplying pixel number to the mean pixel intensity, after appropriate thresholding. TOPRO-3 fluorescence was determined by the number of pixels with fluorescence above the threshold, which was proportional to nuclei number. For each tumor (three animals per group, three randomly selected images each), values were reported as normalized total fluorescence (total fluorescence (LC3/Ki67/TUNEL)/total nuclear fluorescence) and expressed as fold change relative to the mean value obtained from control animals, from two independent staining analyses. The mRFP fluorescence was analyzed using ImageJ (public domain licence, http://imagej.nih.gov/ij/); data are total pixel intensities/cell minus the mean background fluorescence of nuclei. Homogeneity of variances was checked using Levene’s test, and if variances were equal (normalized data or after log transformation) data were analyzed by drug treatment using Student’s t-test or one-way ANOVA with planned contrasts (LC3 expression) or Tukey’s post hoc test for multiple pair-wise comparisons. Where variances were not equal, Welch ANOVA test was used with Games–Howell post hoc tests for pair-wise comparisons (SPSS Statistics 20, SPSS, IBM, Portsmouth, UK).

Top

Conflict of interest

The authors state no conflict of interest.

Saturday, July 18, 2015

Marijuana Helps Heal Broken Bones

By Rob Quinn,  Newser Staff

Posted Jul 17, 2015 7:17 AM CDT 
 
(Newser) – There's a new and very promising use for marijuana, or at least a compound found in it. Researchers discovered that cannabidiol (CBD) worked wonders for rats with broken limbs. When rats with mid-femoral fractures were given CBD, the healing process was "markedly enhanced" within just eight weeks, according to the Israeli researchers, whose work is published in the Journal of Bone and Mineral Research. The researchers say the healing properties of CBD remained even when the non-psychotropic compound was separated from THC, the component that gets people high, the Times of Israel reports.
And the treatment did more than just mend bones: The compound made bones stronger during healing (scientifically, CBD enhanced "the maturation of the collagenous matrix, which provides the basis for new mineralization of bone tissue"). That means the limb "will be harder to break in the future," the researchers say in a press release. "The clinical potential of cannabinoid-related compounds is simply undeniable at this point," lead researcher Dr. Yankel Gabet says. "While there is still a lot of work to be done to develop appropriate therapies, it is clear that it is possible to detach a clinical therapy objective from the psychoactivity of cannabis." (In other marijuana news, pot smokers hit a surprising change at 21.)

Tuesday, July 7, 2015

Doctor facing 175 year sentence for falsely diagnosing roughly 533 healthy people with cancer and forcing them to receive chemotherapy, only to 'line his pockets' with money.

http://insider.foxnews.com/2015/07/07/doctor-farid-fata-be-sentenced-giving-chemo-healthy-patients

Prosecutors are seeking a 175-year sentence for a Michigan doctor who intentionally misdiagnosed hundreds of healthy patients with cancer to line his pockets.
A sentencing hearing began Monday for Dr. Farid Fata, who gave unnecessary cancer treatments to 553 patients. Fata was arrested back in 2013 for the health care fraud.
“There is no real justice,” victim Geraldine Parkin said, adding that she hopes her anger and frustration will finally go away once Fata is sentenced.

Horrifying Claims Against Cancer Doctor Accused of $35M in Medicare Fraud

Arthur Aidala said this morning on “Fox and Friends” that getting chemo when you don’t have cancer “destroys your insides.”
He said that the lesson to be learned here is to ask to see lab reports and paperwork.
Dr. Keith Ablow added that this case proves that doctors aren’t good enough at supervising other doctors.

Friday, July 3, 2015

It's Official: Marijuana Is Medicine

A series of papers in the Journal of the American Medical Association is starting to correct the shameful legacy of drug war politics over cannabis science. But a research catch-22 persists.

by

The nation's top medical organization released a major series of papers on medical cannabis last week in the Journal of the American Medical Association, in a move that constitutes a small step for the AMA, but a giant leap in cannabis medical history.
 
 In five key papers, teams of researchers systematically reviewed dozens of clinical studies of marijuana, speaking in clear language that the "use of marijuana for chronic pain, neuropathic pain, and spasticity due to multiple sclerosis is supported by high-quality evidence."
The review validated what doctors and patients in California have risked their freedom to say for twenty years. The findings also directly refute critics who maintain that "marijuana is not medicine."
"They concluded cannabis is useful," said Dr. Frank Lucido, a Berkeley physician who specializes in cannabis. "I don't think a single study didn't show benefit. ... I think it was very positive."
"It is somewhat affirming to see this come out," said Clint Werner, best-selling author of Marijuana: Gateway to Health. "It's invalidating that talking point that 'it's just a Cheech and Chong show.'"
"What's driving this is a tremendous cultural shift that's preceding the political shift," said Martin Lee, of author Smoke Signals. The AMA is acknowledging "what's been known for 5,000 years."
The AMA actually opposed federal cannabis prohibition in 1937. After losing that round, most doctors have toed the Drug War line ever since. "It's great that they have finally acknowledged there's some medicinal value in cannabis, but the whole thing is so pathetic," Lee said.
Most major news media outlets, however, have spun the JAMA papers negatively, embracing the narrative that many uses for medical pot are still based on poor science. News reports noted that of one of the JAMA studies found that "there was low-quality evidence suggesting that cannabinoids were associated with improvements in nausea and vomiting due to chemotherapy, weight gain in HIV infection, sleep disorders, and Tourette syndrome."
News outlets also pointed to another review that found that "there is some evidence to support the use of marijuana for nausea and vomiting related to chemotherapy, specific pain syndromes, and spasticity from multiple sclerosis. However, for most other indications that qualify by state law for use of medical marijuana, such as hepatitis C, Crohn disease, Parkinson disease, or Tourette syndrome, the evidence supporting its use is of poor quality."
Cannabis experts say the problem with some of the conclusions in the JAMA studies, and the reporting about them, is that they fail to own up to the main reason why study quality has often been poor: the systematic blockade on pot research. For decades, the federal government has refused to authorize research on the medical benefits of cannabis. As a result, the inconclusiveness of some of the research is more a reflection of the federal ban than of the medical effectiveness of pot. "You know how incredibly hard it is to do research that is intended to confirm benefits of cannabis?" said Warner. "It's impossible. We still have a huge catch-22."
Experts also say that the decision by AMA researchers to ignore the research blockade shows their bias. "It's a national embarrassment. The federal designation that cannabis has no medical value is like saying the moon is made of green cheese," Lee said. "It seems the AMA can't say, 'No, the moon is not made of green cheese.'"
The AMA researchers also fail to acknowledge the real world benefits reported by patients. For example, about one in twenty California adults (1.4 million) have used medical cannabis for a "serious" condition and 92 percent of them have reported that it worked. "We have plenty of evidence that it helps for a lot of things," Lucido said. "We should always do more research. But we shouldn't stop people from using it in the meantime."
The AMA researchers also listed the side effects of cannabis without providing context. "Adverse Effects included dizziness, dry mouth, nausea, fatigue, somnolence, euphoria, vomiting, disorientation, and hallucination," one of the reports concluded.
But the AMA researchers didn't compare the adverse side effects of cannabis to those of competing analgesics and anti-inflammatories. Painkiller overdoses are America's leading epidemic. Ibuprofen, for example, regularly causes kidney failure. But those facts were not mentioned in the JAMA studies. "A full third of the Physician's Desk Reference is adverse effects, including death," Lucido said. "Cannabis has about ten adverse effects, at least two of which are often desired."
The AMA researchers' statements about cannabis addiction also lacked context. "I imagine sleeping pills probably have a much bigger dependency ratio," Lucido said.
Still, as half-hearted and equivocal as the JAMA papers were, they contributed to a rising tide of mainstream validation for cannabis that's washing away its prohibition.
"Culturally, the fight is over. We won," Lee said. "The pro-cannabis side has conquered the culture. Now politics is catching up."

http://m.eastbayexpress.com/oakland/its-official-marijuana-is-medicine/Content?oid=4381482