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Kawamura Rumi

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12.09.2018

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  • Kawamura Rumi
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    CBD treatment was initiated upon first detection of the primary tumours 1 week until the completion of the study approximately 1 month. Treatment and analysis were performed as previously described McAllister et al.

    The mouse 4T1 i. One or 7 days after the injection, the tumour-bearing mice were i. Two or 18 days after the injection, the tumour-bearing mice were i. Fifteen minutes before imaging, mice were i. Western blotting was performed as previously described McAllister et al. The blots were probed with anti-Id1 or anti-Id2 McAllister et al. The relative amounts of protein were quantified using densitometry and the software programme ImageJ NIH.

    All values in separate experiments were normalized to the corresponding vehicle control values. Cells were grown in 6-well culture dishes and treated with the appropriate compounds every 24 h for 2 days.

    Labelled cells were analysed by cell flow cytometry using the Muse cell analyzer. Survival between groups was compared using a log-rank Mantel—Cox test.

    Additional methods are described in the Supporting Information. While CBD has been shown to inhibit breast cancer metastasis in vivo , a detailed pharmacological analysis to determine potency and efficacy has not been performed. Utilizing the 4T1 i. The potency of CBD at targeting metastasis in the i. CBD was also effective at reducing metastatic progression in an orthotopic model utilizing 4T1 cells Supporting Information Fig. S2A—C even when the drug was administered three times a week as opposed to daily, but the cannabinoid did not inhibit primary tumour growth.

    In both the i. Since orthotopic models suffer from significant variability and CBD did not inhibit primary tumour growth, we continued our investigations into the anti-metastatic activity of CBD using the i. In the culture, the ability of CBD to inhibit breast cancer aggressiveness is the direct result of down-regulation of Id1 expression McAllister et al.

    To determine whether the anti-metastatic activity of CBD was directly related to the down-regulation of Id1 in vivo , we first determined whether CBD down-regulated Id1 gene expression in the tumour tissue.

    We have previously shown that CBD does not inhibit Id1 expression and invasion in these cells in the culture McAllister et al. Inhibition of Id1 expression in vivo is necessary for the anti-metastatic activity of CBD. A Immunohistochemical detection of Id1 and Ki67 was performed in lung tissues of vehicle left and CBD right treated 4T1-derived tumours.

    Nuclei are visible in blue haematoxylin staining. B The intensity of Id1 expression is shown. C The percentage of Ki67 positive cells per lung metastatic foci was evaluated. Two days after the injection, the tumour bearing mice were injected i. We found that CBD dose-dependently reduced the growth of established lung metastatic foci, reduced the formation of new metastatic foci and increased survival Figure 2B—D. Based upon these findings, we expected that synthesis of more active analogues based upon CBD would result in the development of a compound that could produce more robust inhibition of advanced stages of metastasis.

    CBD reduces the formation of metastatic foci and increases survival in advanced stages of metastatic progression. A The pictures are representative of tumour formation observed at days 5, 7 and 9.

    B Seven days after the injection of tumour cells, mice were injected i. Our past studies McAllister et al. S4A and B suggested the unique activity inhibition of Id1 gene expression of CBD was related to the opened pyran ring, the possession of an extended alkyl side chain and was not due to interactions with the abnormal CBD receptor.

    While selective activation of CB 2 receptors leads to anti-tumour activity Blazquez et al. We reasoned however that a CB 2 selective cannabinoid agonist, having limited activity at CB 1 receptors psychoactivity , could be developed to target Id1, resulting in a single compound that could efficiently target multiple pathways associated with cannabinoid activity leading to enhanced anti-tumour activity. Experiments were carried out as previously described McAllister et al.

    Data represent the mean with corresponding confidence limits for three to six independent determinations. O was previously synthesized in a series of bicyclic resorcinol derivatives that resembled CBD Supporting Information Fig. S4A Wiley et al. It has been previously shown to have lower affinity for CB 1 receptors compared with THC and produces little activity in the tetrad assay measure of psychoactivity in vivo Wiley et al.

    In all studies, the drugs were co-administered. The antago nist alone had no significant effects on cell viability Supporting Information Fig. The ability of the cells to migrate and invade in modified Boyden chambers was then determined.

    Id2 expression was also determined in 4T1 cells treated with vehicle control , 1. While no reversal of CBD activity was observed using the CB 2 receptor antagonist SR, it was able to partially reverse the inhibitory effects of O Id2 is a marker of good prognosis in breast cancer patients and is specifically up-regulated following inhibition of Id1 expression Itahana et al.

    S5C is a primary mechanism that leads to the inhibition of Id1 expression, cell growth, invasion and survival across multiple cancers Ligresti et al.

    D A representative example of the Western blot analysis for LC3 is shown. E The number of cells positive for annexin staining after 2 days treatment with 1. A primary mechanism for the anti-tumour activity of the mixed CB 1 and CB 2 receptor agonist THC and CB 2 selective agonists is the up-regulation of the autophagy pathway Velasco et al.

    We found that O was 2. Moreover, no overt toxicity was noted with O in the mouse models of metastasis as assessed by weight, appearance and general activity data not shown. O is more potent than CBD at inhibiting breast cancer metastasis. One day after the injection, the tumour-bearing mice were i. In agreement with our findings in the culture, the anti-metastatic activity of CBD was not affected by co-administration with SR SR2 , whereas the anti-metastatic activity of O was partially reversed by the antagonist.

    Seven days after i. O produces a significant inhibition of advanced stage breast metastasis. A, B One week after the injection of 4T1 cells, the tumour-bearing mice were injected i. Indicates where one animal responding well to treatment with O based on BLI was removed in order to stain and visualize lung metastatic foci.

    We next utilized MDA-MBluc-D3H2LN cells in order to determine whether O would produce robust anti-metastatic activity against a human breast cancer cell line in advanced stages of disease progression. This variant is significantly more aggressive than the parental line with a rapid disease progression with a time course more closely resembling the 4T1 model.

    Additionally, the expression of luciferase allowed us to assess the activity of the drug treatments longitudinally in real time using BLI and to demonstrate that metastases were present in the lung when the treatment was initiated. This cell line was therefore significantly less sensitive to the effects of CBD but not O While regression did occur beyond the initial size of the tumour in some mice, the tumour did adapt over time and began to progress again.

    One mouse, where the tumour was regressing in the lung, was removed from the study to confirm the imaging results by visualizing the lung using an India ink stain and a dissecting microscope Supporting Information Fig. We therefore initiated treatment with CBD 2 days after mice were i. While CBD inhibited disease progression in a subset of the mice, overall survival was not significantly improved Supporting Information Fig.

    CBD has been reported to have a wide variety of therapeutic indications and has been shown to be non-toxic, safe and well-tolerated in clinical trials Zuardi, One of the most exciting recent areas of study for the therapeutic application of CBD resides in its ability to decrease cancer cell invasion and metastasis Ligresti et al. In this investigation, we determined that CBD was effective at inhibiting metastatic progression, leading to prolonged survival in multiple preclinical models of breast cancer.

    Id1 has been shown to play a key role in mediating breast cancer tumourigenicity and metastasis to the lung Fong et al. We previously reported that CBD could down-regulate Id1 gene expression in breast cancer cell lines in culture McAllister et al. Here, we demonstrated that treatment with CBD can also lead to the inhibition of Id1 gene expression and tumour cell proliferation in lung metastatic foci in a mouse model of breast cancer.

    In addition, ectopic expression of Id1 in breast cancer cells reversed the anti-metastatic activity of CBD. Overall, these data suggest that the anti-metastatic activity of CBD is directly related to the down-regulation of Id1 gene expression; Id1 therefore represents a potential biomarker for predicting whether CBD will be effective at inhibiting tumour progression.

    Additional mechanisms in vivo that have been implicated in the anti-metastatic activities of CBD include the up-regulation of intercellular adhesion molecule-1 and tissue inhibitor of matrix metalloproteinases-1 in lung cancer Ramer et al. In contrast to the moderate doses of CBD needed to inhibit metastatic progression, higher doses of CBD have been required to inhibit tumour growth after s.

    In our previous work, daily administration of CBD only produced a minor delay in primary tumour growth in a model where 4T1 cells where s. In the present manuscript, we observed no inhibition of primary tumour growth in the orthotopic model where the cells where injected into the MFP and when the mice were treated three times a week with CBD.

    Taken together, the ability of CBD to inhibit primary tumour growth at the doses evaluated in our current investigation is limited.

    The differences in the potency of CBD for targeting processes involved in cancer cell growth and survival versus invasion and metastasis may explain why the cannabinoid is less efficient at inhibiting primary tumour growth.

    We noted that, in both the orthotopic and the i. This led us to hypothesize that CBD could be effective at inhibiting the growth of secondary tumours even after their initial establishment in the lung.

    While CBD was effective at inhibiting metastatic progression even in more advanced stages of the disease, it was not efficacious enough to produce substantial increases in survival at this stage. We therefore underwent a screening strategy of cannabinoid analogues focused on inhibition of Id1, cell proliferation and invasion.

    We therefore further envisioned a cannabinoid analogue that could target Id1 and activate cannabinoid receptors. This effect can be reproduced using CB 2 selective agonists, which is an advantage since activation of CB 1 receptors leads to psychotropic effects Salazar et al. We hypothesized that targeting of Id1 expression and cannabinoids receptors with a single compound would result in an even more robust inhibition of advanced stages of metastasis.

    Screening a library of resorcinol derivatives, we discovered a cannabinoid analogue O that could activate CB 2 receptors and was more potent at inducing the formation of ROS and inhibiting Id1 expression in comparison to CBD.

    This activity was not related to the activation of CB 1 , CB 2 or vanilloid receptor 1 similar to the results reported previously in human breast cancer cells Ligresti et al. Taken together, these studies suggest the existence of a unique intracellular interaction site for CBD that regulates calcium homeostasis, leading to generation of ROS. This activity may be one of the initial events leading to the downstream anti-tumour activity of CBD. In addition to inhibiting Id1 expression, the resorcinol derivate O was also found to be an efficacious CB 2 selective agonist.

    The ability of O to inhibit breast cancer aggressiveness in the culture and in vivo was partially reversed by a CB 2 receptor antagonist. CBD did not efficiently activate autophagy and did not induce apoptosis in cell culture at concentrations that produce ROS and target Id1 gene expression. A recent study showed CBD increased autophagy-mediated cell death in breast cancer cells in the culture Shrivastava et al.

    However, the concentration of CBD used in the culture to produce this effect was significantly higher approximately three times than that needed to target Id1 gene expression in our investigations. CBD was also shown to be ineffective at inducing autophagy in vivo when targeting glioblastoma Torres et al. O was significantly more potent and efficacious than CBD in multiple preclinical models of breast cancer. O also produced a significant increase in survival in advanced stages of mouse and human breast cancer metastasis, and in certain instances, O produced regression of established metastatic foci.

    We propose that this is the result of efficiently targeting cannabinoid anti-tumour pathways that have been associated with the activity of both CBD and THC.

    In agreement with this hypothesis, the combined administration of CBD and THC produced a similar magnitude of anti-metastatic activity when compared with O alone. O, however, is significantly less potent than THC at targeting CB 1 receptors and demonstrated limited activity in in vivo assays that predict the potential psychotropic activity of cannabinoids Wiley et al. In addition to the direct anti-tumour activity of cannabinoids, the targeting of Id1 expression and autophagy has been shown to result in the enhanced the activity of first-line agents Hu et al.

    Similar to what was accomplished in this investigation using the Id1 gene as a target for CBD, if primary mechanisms for the beneficial effect of CBD in non-cancer-related diseases could be identified, then there is the potential to develop more potent analogues that retain the activity of THC and CBD. Overall, this study suggests that the use of cannabinoid compounds may represent a potential approach for the treatment of patients with metastatic breast cancer and provides a framework for the synthesis of additional novel cannabinoid analogues based on our lead compound.

    All authors have made substantial contributions to the conception and design of the study, or acquisition of data, or analysis and interpretation of data, or drafting of the manuscript. Figure S1 CBD produces a dose-dependent reduction of metastatic spread to the lung and increases survival. A One day after the injection, the tumour-bearing mice were injected i. November 22, Volume 24 Issue 2 Pages A surgical case of superficial esophageal basaloid cell carcinoma.

    August 30, Volume 24 Issue 2 Pages Already have an account? Annals of Cancer Research and Therapy. Journal home Journal issue About the journal. Most viewed articles Jan.

    A review of clinical and molecular evidence Released: A study protocol Released: Present situation on Cancer Released:

    Creator Information

    The latest Tweets from Rumi Kawamura (@KawamuraRumi). Rumi Kawamura's 3 research works with citations and reads, including: Suppression of invasion and metastasis in aggressive salivary cancer cells. Rumi Kawamura's 3 research works with 7 citations and 71 reads, including: Early‐stage diffuse panbronchiolitis in a young patient confirmed by video‐ assisted.

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    Comments

    truestars2009

    The latest Tweets from Rumi Kawamura (@KawamuraRumi).

    PRO100KOT

    Rumi Kawamura's 3 research works with citations and reads, including: Suppression of invasion and metastasis in aggressive salivary cancer cells.

    geos9127

    Rumi Kawamura's 3 research works with 7 citations and 71 reads, including: Early‐stage diffuse panbronchiolitis in a young patient confirmed by video‐ assisted.

    Karam

    Tokyo Arts and Space is an art center dedicated to the generation and promotion of new art and culture from Tokyo.

    drlektor1

    Rumi Kawamura - Google+. Rumi Kawamura. 2 followers. 2 followers. About. Posts. Looks like you've reached the end. Looks like you've reached the end.

    sergiyze

    Liliana Soroceanu, Ryuichi Murase, Chandani Limbad, Eric Singer, Juanita Allison, Isabel Adrados, Rumi Kawamura, Arash Pakdel, Yayoi Fukuyo, Daniel.

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