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Medical Marijuana Risks Kaposi's Sarcoma
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Author Comments: "....These results suggest that low doses of 9-THC may foster the initiation of Kaposi's sarcoma and contribute to its progression and spread..... Our studies strongly suggest that 9-THC could worsen KSHV infection and foster development of the neoplasm.... for the medical use of purified cannabinoids like 9-THC, our study suggests that their administration may place patients at greater risk for KSHV infection and Kaposi's sarcoma and that treatment should be evaluated on a case by case basis under close medical supervision. Further epidemiologic studies and clinical research are needed to clarify the importance and safety of administering cannabinoids like 9-THC.... Cannabinoids are a family of multifunctional compounds and their targets are not fully understood....9-THC may also bind to other unknown receptors.... Further investigation is needed to clarify the multifunctional effects of cannabinoids.
"Marijuana Component Opens the Door for Virus That Causes Kaposi's Sarcoma"
FULL TEXT BELOW
Source: American Association for Cancer Research (AACR)
Wed 01-Aug-2007, 11:10 ET
Description
The major active component of marijuana could aid the Kaposi's sarcoma virus in infecting cells and multiplying, according researchers at Harvard Medical School. In the August 1 issue of Cancer Research, they report that low doses of delta-9 tetrahydrocannabinol (THC), equivalent to that in the bloodstream of an average marijuana smoker, could be enough to facilitate infection of skin cells and could even foster malignancy.
Newswise - The major active component of marijuana could enhance the ability of the virus that causes Kaposi's sarcoma to infect cells and multiply, according to a team of researchers at Harvard Medical School. According to the researchers, low doses of _-9 tetrahydrocannabinol (THC), equivalent to that in the bloodstream of an average marijuana smoker, could be enough to facilitate infection of skin cells and could even coax these cells into malignancy.
While most people are not at risk from Kaposi's sarcoma herpes virus (KSHV), researchers say those with lowered immune systems, such as AIDS patients or transplant recipients, are more susceptible to developing the sarcoma as a result of infection. Their findings, reported in the August 1 issue of Cancer Research, a journal of the American Association for Cancer Research, offer cautionary evidence that those with weakened immune systems should speak with their doctors before using marijuana medicinally or recreationally.
"These findings raise some serious questions about using marijuana, in any form, if you have a weakened immune system," said lead study author Jerome E. Groopman, M.D., professor of medicine at Harvard Medical School. "While THC is best known as the main psychotropic part of marijuana, an analog of THC is the active ingredient of marinol, a drug frequently given to AIDS patients, among others, for increasing appetite and limiting chemotherapy-induced nausea and vomiting."
While previous studies indicated that marijuana smoking was associated with Kaposi's sarcoma, this is the first to demonstrate that THC itself can assist the virus in entering endothelial cells, which comprise skin and related tissue.
According to Dr. Groopman, the study illustrates the complicated role marijuana and other cannabinoids play in human health. Numerous types of cells display cannabinoid receptors on their outer surfaces, which act as switches that control cellular processes. Dr. Groopman's laboratory had previously demonstrated that THC could have a protective effect against a certain form of invasive, drug-resistant lung cancer.
To study the combined effect of THC and KSHV, the researchers examined a culture of human skin cells, which are susceptible to infection and could provide a model of Kaposi's sarcoma. These culture cells display many copies of two prominent cannabinoid receptors. Dr. Groopman and his colleagues found that by bonding to these receptors, low doses of THC activate two proteins responsible for maintaining a cell's internal framework, or cytoskeleton. By altering the cytoskeleton, THC effectively opens the door for KSHV, allowing the virus to more easily enter and infect the cell. "We can take away that effect by using antagonists that block the two cannabinoid receptors, which adds evidence that THC is the culprit," Dr. Groopman said.
Once a cell is infected, the presence of THC may also promote the cellular events that turn it cancerous, the researchers say. They found that THC also promotes the production of a viral receptor similar to one that attracts a cell-signaling protein called interleukin-8. Previous studies have noted that this receptor could trigger the cell to reproduce, causing Kaposi's sarcoma-like lesions in mice. Indeed, the researchers saw that THC induced the infected cells to reproduce and form colonies in culture.
"Here we see both infection and malignancy going on in the presence of THC, offering some serious concerns about the safety of THC among those at risk," Dr. Groopman said. "Of course, we still do not know the exact molecular events that are occurring here, but these results are just the first part of our ongoing research."
The study was funded by the National Institutes of Health.
Cannabinoid Modulation of Kaposi's Sarcoma-Associated Herpesvirus Infection and Transformation
Cancer Research 67, 7230-7237, August 1, 2007
Xuefeng Zhang1, Jian Feng Wang1, George Kunos3 and Jerome E. Groopman1,2
1 Division of Experimental Medicine and 2 Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts and 3 National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, Maryland
Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV; also named human herpesvirus 8) is necessary but not sufficient for the development of Kaposi's sarcoma. A variety of factors may contribute to the pathogenesis of Kaposi's sarcoma in addition to KSHV. Marijuana is a widely used recreational agent, and 9-tetrahydrocannabinol (9-THC), the major active component of marijuana, is prescribed for medicinal use. To evaluate how cannabinoids may affect the pathogenesis of Kaposi's sarcoma, we studied primary human dermal microvascular endothelial cells (HMVEC) exposed to KSHV. There was an increased efficiency of KSHV infection in the presence of low doses of 9-THC. We also found that 9-THC increased the viral load in KSHV-infected HMVEC through activation of the KSHV lytic switch gene, the open reading frame 50. Furthermore, we observed that 9-THC stimulated expression of the KSHV-encoded viral G protein-coupled receptor and Kaposi's sarcoma cell proliferation. Our results indicate that 9-THC can enhance KSHV infection and replication and foster KSHV-mediated endothelial transformation. Thus, use of cannabinoids may place individuals at greater risk for the development and progression of Kaposi's sarcoma.
Introduction
Kaposi's sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi's sarcoma, is not sufficient for the development of Kaposi's sarcoma. This is highlighted by the observation that most KSHV infections are asymptomatic, and >95% of persons who become infected never develop Kaposi's sarcoma or other KSHV-related cancers (1, 2). It seems that certain cofactors participate in the development of Kaposi's sarcoma mainly through suppression of the immune system (3, 4). Whereas the immune system of healthy adults usually holds the virus in check, AIDS patients (5), transplant recipients (6), and other immunocompromised patients are at risk for Kaposi's sarcoma (7) if they are KSHV infected.
Marijuana, the prototypical cannabinoid, has been used as a recreational, ceremonial, and therapeutic substance throughout history. Although cannabinoids display a series of promising therapeutic potentials (8, 9), marijuana and other cannabinoids may impair both cell-mediated and humoral immune system function, leading to decreased resistance to infection by viruses and bacteria (10-12). Hence, marijuana smoking has been postulated to act as a possible cofactor in the development of Kaposi's sarcoma, as it predisposes human immunodeficiency virus-positive individuals to opportunistic infections and Kaposi's sarcoma (13-15). A previous study showed that 9-tetrahydrocannabinol (9-THC) inhibited the lytic replication of KSHV in B cells by modulating the expression of open reading frame 50 (ORF50; ref. 16). However, these results cannot be extrapolated to the case of Kaposi's sarcoma because its lesions are mainly composed of spindle cells, which seem to be of lymphatic endothelial origin (17). Furthermore, the transcriptional profiling of KSHV genes is different in endothelial cells and B cells (18). Thus, the effects of cannabinoids on KSHV are not yet characterized. 9-THC is highly lipophilic and can change membrane permeability (19), suggesting that this cannabinoid could modulate KSHV entry into the endothelium. Moreover, 9-THC initiates a signaling cascade that could modulate cellular susceptibility to KSHV and alter the viral life cycle, as well as promote viral strategies of immune evasion. For these reasons, the potential risks versus benefits of cannabinoids need to be explored in KSHV-associated diseases.
Here, we report that low doses of 9-THC, similar to those achieved in vivo, facilitated KSHV infection in endothelial cells through enhancement of cell-cell interactions and endocytosis. We further show that 9-THC up-regulated the expression of both the lytic switch gene ORF50 and the carcinogenic KSHV G protein-coupled receptor (GPCR), thereby increasing viral titers in culture and inducing endothelial cell transformation. These results suggest that low doses of 9-THC may foster the initiation of Kaposi's sarcoma and contribute to its progression and spread.
Discussion
KSHV infection is prevalent and lifelong, and identification of cofactors that may contribute to the pathogenesis of Kaposi's sarcoma could assist in its prevention (28). Based on epidemiologic studies, drug addiction increases the risk of acquiring infectious diseases (15). In addition, marijuana use is common in U.S. and European populations, and medicinal treatment with cannabinoids is increasing. Thus, in this report, we address the role of cannabinoids in the pathogenesis of Kaposi's sarcoma. Our studies strongly suggest that 9-THC could worsen KSHV infection and foster development of the neoplasm.
We found that 9-THC modulated host-virus interactions via its effects on endocytosis. We also observed that low doses of 9-THC (<1 μmol/L) activated the cytoskeletal system in HMVEC, with significant phosphorylation of p130Cas and paxillin, two critical components of the focal adhesion complex. These proteins are required for the control of actin organization and the formation of microvilli-like cellular protrusions and for the internalization of bacteria as well as the migratory responses of cells (29-31). In particular, p130Cas protein has been reported to participate in the uptake of adenovirus, human respiratory syncytial virus, and Yersinia through either endocytosis or phagocytosis (32-34). Furthermore, both p130Cas and paxillin proteins interact with focal adhesion kinase (FAK) and are tyrosine phosphorylated during integrin-mediated adhesion. Integrins and FAK are involved in KSHV entry into target cells (21, 35). Our findings indicate that cannabinoids like 9-THC may facilitate KSHV infection through activation of certain cytoskeletal proteins.
We further showed that 9-THC modified KSHV status through regulation of the key viral gene ORF50. Maintenance of latent status and reactivation of lytic replication are essential for Kaposi's sarcoma occurrence. The latent infection contributes to immune evasion, whereas lytic infection leads to the production of infectious viral progeny. KSHV latency usually dominates the KSHV life cycle, and KSHV starts its lytic cycle through unclear mechanisms. Because ORF50 expression is sufficient for KSHV lytic viral reactivation and ORF50 targeting efficiently inhibits KSHV replication, it has been recognized as a major switch gene for KSHV reactivation (36, 37). In prior studies, TPA and hypoxia were found to stimulate the lytic replication of KSHV via ORF50, and methotrexate was shown to inhibit TPA-mediated lytic replication also through ORF50 (38-40). Therefore, 9-THC-induced ORF50 gene expression and subsequent KSHV lytic replication may significantly change the course of KSHV-associated diseases.
Moreover, we showed that 9-THC strongly induced the expression of KSHV GPCR on the cell surface and significantly fostered endothelial transformation in vitro. Kaposi's sarcoma is a typical vascular tumor expressing high levels of VEGF and VEGFR. 9-THC has been reported to inhibit the angiogenesis and tumorigenesis of gliomas via the VEGF/VEGFR pathway (41). However, this does not seem to be the case in Kaposi's sarcoma because the dose-dependent inhibitory effects of 9-THC on VEGF/VEGFR were only observed in HMVEC and not in KSHV-infected HMVEC and Kaposi's sarcoma cells (data not shown). In contrast, we found that 9-THC induced the expression of KSHV GPCR, a homologue of the IL-8 receptor. It has been reported that constitutively active KSHV GPCR triggers the autonomous proliferation of endothelial cells, causes Kaposi's sarcoma-like lesions in mice, and initiates the development and progression of Kaposi's sarcoma (42-45). The differing effects of 9-THC on KSHV observed in our study and other reports may be due to the varied transcriptional activation of KSHV genes in different cell types (endothelial cells versus B cells) after infection. In addition, different concentration ranges of 9-THC (low versus high dosage) as well as varied technological approaches were used in our study as compared with the previous report. Hence, it seems that distinct pathogenetic pathways may exist for KSHV-associated neoplasms.
Cannabinoids are a family of multifunctional compounds and their targets are not fully understood. It is known that 9-THC binds to the CB1 and CB2 receptors with equal affinity. However, 9-THC may also bind to other unknown receptors because vanilloid receptor 1 and GPCR55 also serve as cannabinoid receptors (46, 47). Here, we found that both CB1 and CB2 receptors as well as an endothelial cannabinoid receptor distinct from CB1 and CB2 (20) may be involved in the 9-THC-mediated effects on KSHV because selective antagonists for each of these receptors only partially blocked the 9-THC-induced effects. Further investigation is needed to clarify the multifunctional effects of cannabinoids.
In conclusion, our study indicates that 9-THC in concentrations easily achieved in vivo may enhance KSHV infection, accelerate KSHV replication, and/or foster KSHV-mediated transformation. These observations suggest that 9-THC may accelerate the development, progression, and spread of Kaposi's sarcoma. It is important to emphasize that marijuana is not a single drug but a complex botanical substance. Although 9-THC is the major psychoactive component of marijuana, many of its other components are not well elucidated. The clinical pharmacology of the constituents of marijuana is likely complicated, particularly when the plant is smoked or eaten in its natural form. Indeed, both viral enhancers and inhibitors may coexist within marijuana. Hence, for the medical use of purified cannabinoids like 9-THC, our study suggests that their administration may place patients at greater risk for KSHV infection and Kaposi's sarcoma and that treatment should be evaluated on a case by case basis under close medical supervision. Further epidemiologic studies and clinical research are needed to clarify the importance and safety of administering cannabinoids like 9-THC.
Results
Low doses of 9-THC increase KSHV infection. We first observed robust expression of the cannabinoid receptors, CB1 and CB2, in HMVEC (Fig. 1A ). Next, to investigate whether 9-THC affects KSHV infection, HMVEC were treated with different doses of 9-THC (0, 0.001, 0.01, 0.1, and 1 μmol/L) or vehicle control for 1 h before exposure to GFP-KSHV. Low doses of 9-THC (<1 μmol/L) increased KSHV infection in a concentration-dependent manner compared with the vehicle treatment (Fig. 1B and C). High doses of 9-THC (>1 μmol/L) caused considerable cell death in combination with KSHV exposure (data not shown). In addition, we found that KSHV infection of endothelial cells slightly up-regulated the expression of CB1 and CB2 (data not shown). However, we did not observe any significant changes in the expression of integrin a3é1, vascular endothelial growth factor (VEGF) receptor (VEGFR)-3, or DC-SIGN on treatment with 9-THC (data not shown).
We next sought to determine whether 9-THC may modulate KSHV entry into endothelial cells. KSHV mainly enters target cells through endocytosis, which is controlled by reorganization and rearrangement of the cytoskeleton (21, 24). To this end, we studied the effects of 9-THC on the cytoskeletal system. By using Western blot analysis, we found that two important cytoskeletal proteins, p130Cas and paxillin, were significantly phosphorylated after exposure to 9-THC (Fig. 2A ). To further confirm the above observations, we did confocal scanning to detect activated paxillin under the same conditions. As shown in Fig. 2B, activated paxillin exhibited staining with fluorescein. The basal level of activated paxillin was observed to be uniform in the endothelial cytoplasm in quiescent cells. After 9-THC stimulation, significantly more paxillin was activated and aggregated beneath the cell surface. Cell membranes seemed invaginated and some pseudopodia-like dendrites were formed. These morphologic changes in cell shape can increase the interaction of cells with viruses, enhance endocytosis, and could also account for the observed increase in KSHV infection. In this regard, a specific endocytosis inhibitor, cytochalasin D, was used to block the activation of the cytoskeletal system induced by 9-THC. Pretreatment with cytochalasin D (1 μmol/L; Sigma-Aldrich) significantly decreased KSHV infection in the presence of 9-THC (Fig. 2C), suggesting that 9-THC modulates the process of endocytosis and leads to higher KSHV infection efficiency in HMVEC.
Low doses of 9-THC induce KSHV replication in endothelial cells. The status of the KSHV life cycle is closely linked to the course of KSHV-associated diseases. The major latency-associated nuclear antigen of KSHV (LANA-1, ORF73) is expressed by infected cells and has been commonly used as a marker of KSHV load in real-time PCR assays (23). Chemical agents like 12-O-tetradecanoylphorbol-13-acetate (TPA) induce KSHV and have been used as a positive control to study cannabinoid effects on viral latency. We found a significant increase in the number of LANA-1 copies in KSHV-infected HMVEC on treatment with 9-THC but not in the culture supernatants (Fig. 3A and C ). Viral loads were observed to increase over time within a week after exposure to 9-THC (data not shown). Basal titers of KSHV in the HMVEC were much lower than those in the GFP-BCBL-1 cells. However, the effect of 9-THC on viral titers was more significant in the KSHV-HMVEC than in the GFP-BCBL-1 cells (data not shown). These results suggest that the KSHV life cycle is cell dependent and is differentially modulated in response to certain stimuli like cannabinoids.
We then assessed the expression of ORF50, the major switch gene for KSHV from latency to the lytic cycle, after treatment of endothelial cells with 9-THC. We observed that ORF50 expression was up-regulated by 9-THC in KSHV-infected HMVEC (Fig. 3B). These results indicate that 9-THC-induced ORF50 expression can promote KSHV lytic replication in HMVEC.
Low doses of 9-THC foster KSHV-infected HMVEC to form colonies in vitro. 9-THC has been reported to enhance tumor cell proliferation and to accelerate cancer progression (25-27). We found a biphasic activity of 9-THC on KSHV-mediated transformation. Low doses of 9-THC stimulated colony formation, whereas high doses of 9-THC inhibited colony formation (Fig. 4A ). To decipher how 9-THC may change the growth status of tumor cells, we investigated the expression levels of KSHV GPCR, one of the key elements in KSHV-mediated cell transformation. We observed a pattern of KSHV GPCR distribution on the cell surface similar to that of normal GPCRs. Isolated KSHV GPCR pits or vesicles were observed on the cell membrane in KSHV-infected HMVEC in the absence of cannabinoid treatment. However, significantly more and brighter KSHV GPCR vesicles aggregated on the cell surface of spindle-shaped cells after exposure to 9-THC (Fig. 4B). The increased expression of KSHV GPCR was further confirmed by Western blotting (Fig. 4C). These results suggest the involvement of KSHV GPCR in the transforming effects of 9-THC.
To show whether KSHV GPCR is integrally involved in 9-THC-induced cell proliferation, we transiently transfected HMVEC with plasmids encoding either KSHV GPCR or vector alone and then quantitated and compared the effect of 9-THC on cell proliferation. We found a significant increase in cell proliferation following 9-THC treatment of the KSHV GPCR-expressing cells but no such effect with the control cells (Fig. 4D). In addition, we observed no significant changes in growth-regulated oncogene- or interleukin-8 (IL-8) production in the cultured KSHV-infected HMVEC (data not shown). These two growth factors have been reported to stimulate Kaposi's sarcoma growth through binding to their cognate receptors and KSHV GPCR. These data indicate that neither autocrine nor paracrine loops contribute to the effects of 9-THC, but rather that up-regulation of the constitutively active KSHV GPCR mediates the effects of 9-THC on Kaposi's sarcoma cell proliferation.
Low doses of 9-THC induce the adhesion of KSHV-infected B cells to endothelial cells. Latent-infected lymphocytes constitute the major pathologic reservoir of KSHV in vivo. When KSHV latency switches into lytic replication in B cells and lytically infected lymphocytes circulate or home to adjacent lymph nodes, interactions between lymphocytes and endothelial cells frequently occur, which may enhance viral secondary transmission. To address whether 9-THC could modulate viral transmission, we used an in vitro static adhesion assay to investigate the effects of 9-THC on the interaction between lymphocytes and endothelial cells. Lytically infected GFP-BCBL-1 cells were prepared by using TPA (20 ng/mL) and then coincubated with HMVEC. Both GFP-BCBL-1 cells and HMVEC were treated in the absence or presence of 9-THC, as indicated. We found that 9-THC significantly enhanced the adhesion of GFP-BCBL-1 cells to HMVEC (Fig. 5A ), leading to increased KSHV infection in HMVEC compared with the vehicle control (data not shown). Further experiments showed that the 9-THC-induced adhesion was due to up-regulated PECAM-1 expression (Fig. 5B), as neutralized anti-PECAM-1 antibody blocked the 9-THC-induced effects (Fig. 5C). These data suggest that 9-THC stimulates expression of the endothelial-specific focal adhesion molecule PECAM-1, resulting in enhanced cell-cell interactions.
Different cannabinoid receptors are involved in 9-THC-mediated signal transduction. To investigate which cannabinoid receptors might play an important role in the 9-THC-induced effects, we blocked cannabinoid receptors with specific antagonists of the receptors. We observed that 9-THC induced significant expression of PECAM-1 and é-catenin but not ICAM-1 in KSHV-infected HMVEC. Specific antagonists, including AM251, AM630, and O-1918, only partially blocked these effects (Fig. 6 ). Our results suggest that different cannabinoid receptors are involved in 9-THC-mediated signal transduction.
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