triple negative breast cancer and cbdDecember 15, 2021
‘Milestone’ as first cancer patients treated with medical cannabis formulations.
“I first started having problems aged 11 and I was diagnosed on my 23rd birthday. I had my first surgery in June 2019, where they removed quite extensive endometriosis,” she says.
However, Harriet’s prescription currently costs her around £300 a month, and for many people living with a long-term health issue, this isn’t feasible.
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TNBC is more likely to affect younger people, those of African descent, Hispanics, and/or people with a BRCA1 gene mutation and is associated with a worse prognosis than other forms of breast cancer.
10th December 2021.
“I started using cannabis a lot more after that surgery because I didn’t like the heavy pharmaceutical drugs that I’ve been offered, which I had awful side effects from.”
She adds: “I’m in a very privileged position financially, and in that I have an incredible relationship with my mother and a boyfriend who understands and advocates for endometriosis, not everyone has that support network. Not everybody is in the financial position to have access to something that could change their life.”
“I think the UK is facing an endometriosis crisis at the moment, particularly as a result of Covid,” says Harriet.
“I was bleeding through all of my clothes, we had to get a new mattress, it was just horrendous,” she says.
Medical cannabis patient, Harriet, on why the UK is facing a crisis in endometriosis care.
“My attendance wasn’t great, I had time off pretty much every time I had my period, I always said I had a cold or food poisoning or something like that,” she says.
Metastatic TNBC is an aggressive form of breast cancer, with limited treatment options.
Earlier this year, third-party testing showed that Apollon’s medical cannabis formulations were “particularly effective” in killing living HER2+ cancer cells, the cause of around 20 percent of all breast cancers.
He added: “The Apollon medical cannabis formulations are expected to be comparatively much less expensive, providing an opportunity for more women to have their triple -negative breast cancer treated.
“I think it comes down to being somebody who has a reproductive health condition. Sometimes I have felt almost a resentment towards patients who have a condition that doesn’t get better.”
Medical cannabis linked to long-term cognitive improvement.
Cancer and medical cannabis: “I saw the positive effects, even after just a few days”
Harriet’s second surgery was delayed almost a year by the coronavirus pandemic, and she ended up having it on New Years Eve of 2020, where it was discovered the endometriosis had spread to her bladder and she was diagnosed with internal cystitis. She is currently recovering from her third surgery in under three years.
Triple-negative breast tumor cells express basal markers, such as epidermal growth factor receptor (EGFR) and cytokeratin 5/6 at high levels. In these tumors, higher expression levels of basal markers, such as EGFR, are associated with a poorer outcome. Although EGFR inhibitors are effective in treating cancer, the early onset of drug resistance limits their therapeutic success . In SUM159 and SCP2 human tumor cells, as model cells for triple-negative breast cancer, CBD effectively inhibited epidermal growth factor (EGF)-induced tumorigenic properties of these cancer cells by obstructing signaling pathways for EGFR, Akt, ERK, and NF-κB. Furthermore, CBD is able to block the secretion of matrix metalloproteinases (MMPs) and the effects of EGF on the cytoskeleton .
Importantly, CBs also bind and activate several other receptors, including the GPRs, GPR18, GPR55, and GPR119. Of particular interest is GPR55, which is activated by lysophospholipid and also by the endocannabinoids AEA and 2-AG. Downstream targets of GPR55 include phospholipase C (PLC), transforming protein RhoA (RhoA), Rho-associated protein kinase (Rock), extracellular-signal-regulated kinase (ERK), and p38 MAPK . CB-Rs form heterodimers with other GPRs, e.g., GPR55, which consequently affects the functions of both receptors. Other GPRs, which are activated by CBs, are acetylcholine receptors and 2-alpha adrenoreceptors as well as opioid-, adenosine-, 5-hydroxytryptophan-, angiotensin-, prostanoid-, dopamine-, melatonin-, and tachykinin receptors. Furthermore, the peroxisome proliferator-activated receptors (PPARs) α and γ are also considered to be receptors for endocannabinoids .
Cannabigerol (CBG) was found to improve digestive functions and has powerful antiemetic and anti-inflammatory effects. CBG is a partial agonist for CB1-R and CB2-R . It may be used for the treatment of neurological disorders.
Cannabis sativa ( C. sativa ) was known among ancient Asian, African, and European agricultural societies. Due to its hallucinogenic effects, Cannabis sativa was applied in religious ceremonies, but it was also widely used in fiber manufacturing, nutrition and medicine. However, in the early part of the last century, C. sativa lost its importance in industry and medicine [1,2]. At present, application of C. sativa in industry and medicine is experiencing a revival. Since 1990, C. sativa became important as a source of compounds to treat cancer and life-threating diseases. The C. sativa plant contains >500 chemical and biologically active compounds . So far, 60 structures have been identified as belonging to the family of cannabinoids (CBs). CBs share a lipid structure featuring alkylresorcinol and monoterpene moieties (terpenophenols) [2,4].
Nabilone (Cesamet ® ) and Dronabinol (Marinol ® ) are synthetic molecules that mimic the pharmacological activity of THC. Their chemical structures are presented in Figure 1 .
4.2. Cannabinoid Receptor Signaling.
Antitumoral activity of CBs in hormone-dependent and –independent breast cancer cell lines.
Resembling the effects of CBD, THC has pro-apoptotic effects in a number of breast cancer cell lines (EVSA-T, MDA-MB-231, MDA-MB-468, SKBR-3, MCF-7 and T-47D) . It reduces cell cycle progression and induces apoptosis in hormone-sensitive and hormone-resistant human breast cancer cell lines. In this way, THC induces cell cycle arrest at the G2/M transition, causing downregulation of Cdc2 and inducing ROS formation to induce cancer cell death. This mechanism is also seen in a number of other cancer cell types e.g., glioma cells .
Mechanism of CB-R-mediated antitumor activity in breast cancer cells. By binding to CB1-R and CB2-R, CBs inhibit breast cancer cell proliferation through various mechanisms. They block cell cycle progression at the G1/S phase via CB1-R and at the G2/M phase via CB2-R activation. They induce breast cancer cell death via apoptosis, mediated by the activation of the transcription factor jun-D. In HER2-overexpressing breast cancer cells, they block cancer cell proliferation in culture and tumors by inhibiting Akt and ERK signaling. They also inhibit cell migration and angiogenesis via CB2-R. CB1-R activation inhibits the FAK/SRC/RhoA pathway leading to inhibition of cell migration. Cell migration blockade is also achieved by CB2-R activation through the inhibition of COX-2 and ERK signaling, which is important for triple-negative breast cancer. AC: adenylate cyclase; Akt: protein kinase B; CB-R: cannabinoid receptor; COX-2: cyclooxygenase-2; EMT: epithelial-mesenchymal transition; ERK: extracellular-signal-regulated kinase; FAK: focal adhesion kinase; GPR: G-protein coupled receptor; HER2: human epidermal growth factor receptor 2: MAPK: mitogen-activated protein kinase; mTOR: mammalian target of rapamycin; PI3K: Phosphoinositol-3-kinase; Raf: serine/threonine-protein kinase; RhoA: transforming protein RhoA; SRC: proto-oncogene tyrosine-protein kinase Src.
At molecular levels, the activation of CB-Rs confers signals of endo, phyto, and synthetic CBs ( Figure 1 ) via inhibition or activation of a variety of signaling pathways  ( Figure 2 ). An important signal transduction pathway regulated by CB-R is linked to the synthesis of ceramide with palmitoyl-transferase as the rate-limiting enzyme in ceramide synthesis . Long-term treatment with ceramide, which activates the proto-oncogene serine/threonine-protein kinase (RAF1), leads to sustained activation of p42/p44 MAPK and induction of apoptosis, as demonstrated in a glioma cell line. This activation could be blocked by CB-R agonists, including THC, by the synthetic CB WIN55,212-2, and the endocannabinoids AEA and 2-AG. However, the duration of the activation of p42/p44 MAPK seems to be critical to the apoptotic response because a protective role of CBs against ceramide-induced apoptosis was also reported .
In 2006, Ligresti et al. demonstrated that CBD caused a potent and selective inhibition of breast cancer cell growth . A number of breast cancer cell lines, such as estrogen receptor (ER)-positive MCF-7, ZR-75-1, and T47D cells, and ER-negative cell lines MDA-MB-231, MDA-MB-468 and SK-BR3, are sensitive to the antiproliferative effects of CBD [27,50,51,52,53]. CBD interferes with cell cycle progression and causes an increase in the number of breast cancer cells in the resting G0 stage and in the G1 compartment. At higher concentrations, CBD causes cell death . Shrivastava et al. showed that in CBD-treated breast cancer cells, a complex interplay between apoptosis and autophagy exists. In the MDA-MB-231 breast cancer cell line, CBD leads to an increase in the generation of ROS, which finally results in an induction of apoptosis and autophagy . Using the MDA-MB-231 breast cancer cell line, it was further shown that beclin 1, a protein that interacts with either B cell lymphoma-2 or PI3K, plays a central role in the induction of autophagy and cell death. CBD causes apoptosis through the production of ROS by changing the mitochondrial permeability transition pore opening, as first demonstrated in human monocytes . CBD inhibits protein kinase B (Akt) and mammalian target of rapamycin (mTOR) signaling and induces autophagy and cell death under oxidative stress conditions. An interplay among decreased mTOR and cyclin D1 together with an upregulated PPARγ expression promotes the induction of apoptosis, a process that is independent of the expression of ERs .
Pharmacological targeting of ERα has been proved to be effective for the prevention and treatment of breast cancer . The majority of newly diagnosed breast cancer (>70%) express ERα and ERβ and are sensitive to estrogen-mediated growth stimulation. Estrogen induces the expression of genes associated with cellular proliferation and survival and contributes to breast cancer development and progression. Importantly, estrogen-sensitive tumors are successfully treated by an antihormonal therapy, and the expression of ERα is a positive prognostic marker for the patient’s risk of a future outcome [43,84]. Also, high levels of ERβ are associated with a better prognosis for the survival . Although CBs do not bind to ERs , THC was found to exert antiestrogenic activities in breast cancer cell lines. Both estrogen and CBs influence pathways associated with cell growth, cell death, and tumor progression, and their antagonistic effects on pathways involving adenylate cyclase, MAPK, ERK, PI3K, and J-Jun may maintain homeostasis between cell survival and cell death . Thereby, the CB-R-induced activation of ERβ-mediated transcriptional activation will disrupt ERα signaling, leading to a reduced expression of estrogen-regulated genes that promote cell growth .
Studies in breast cancer cell lines and animal models showed that an extract from C. sativa was more potent than CBD and THC. Minor CBs in the extract may also contribute to the observed anticancer activity by modulating various targets in the pro-oncogenic pathways leading to an “entourage effect” against cancer cells .
The term ‘endocannabinoid’ was invented in the mid-1990s after the discovery of membrane receptors for THC and their endogenous ligands. It now comprises a whole signaling system consisting of the ‘classical’ CB-Rs, their endogenous ligands, which are lipid signaling molecules called endocannabinoids, and the associated biochemical machinery, including precursor molecules, enzymes for synthesis and degradation, and transporter proteins, such as fatty acid binding protein and heat shock protein 70 . There is now a growing number of endocannabinoid molecules known, which share a similar structure and are natural ligands of the two CB-Rs, CB1-R and CB2-R. They seem to be involved in an increasing number of pathological conditions. Plant-derived CBs (phytocannabinoids, phyto-CBs) as well as synthetic CBs interfere with the endocannabinoid system, and a number of pharmacological effects of phyto-CBs can be explained by this interference .
2-AG: 2-Arachidonoylglycerol; AEA: anandamide; Akt: protein kinase B; AMP: adenosine monophosphate; CBC: cannabichromene; CBD: cannabidiol; CBDA: cannabidiolic acid; CBN: cannabinol; CBG: cannabigerol; CB-R: cannabinoid receptor; Cdc: cell division control; COX: cyclooxygenase; GTP: guanosine triphosphate; IC: inhibitory concentration; ID-1: inhibitor of DNA binding 1; IFN: interferon; IL: interleukin; MAPK: mitogen-activated protein kinase; Met-F-AEA: 2-methyl-2’-F-anandamide; MMP: matrix metalloproteinase; mTOR: mammalian target of rapamycin; Myc: avian virus myelocytomatosis; n.d.: not determined; n.m.: non-malignant; PPAR: peroxisome proliferator-activated receptor; PRLr: prolactin receptor; RAF: proto-oncogene serine/threonine-protein kinase; Rho: transforming protein RhoA; SHARP: SMART/HDAC1 associated repressor protein; TGF: tumor growth factor; Th: T helper; THC: tetrahydrocannabinol; THCA: tetrahydrocannabinolic acid; TIMP: tissue inhibitor of metalloproteinases; trk: tyrosin kinase; TRPV: transient receptor potential cation channels; VEGF: vascular endothelial growth factor.
Cannabinol (CBN) is a non-psychoactive CB with a higher concentration in aged plants, or in degraded or oxidized CB preparations. Pharmacologically relevant quantities are formed as a metabolite of THC. CBN is a partial CB1-R agonist, but it has a higher affinity to CB2-R than to CB1-R.
2. Mechanism of Cannabinoid Action.
Breast cancer is the most frequently diagnosed cancer in women worldwide. There is also an increasing tendency for aggressive subtypes of breast cancer, particularly in women of younger ages [6,42]. Although the main intrinsic molecular subtypes are breast cancer hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative luminal A and B tumors, HER2-enriched tumors and triple-negative tumors, which are usually the most aggressive type, have been identified. As these molecular subtypes differ in the course of the disease and the clinical outcome, individualized therapies will achieve a better outcome for individual patients . Interestingly, data from preclinical in vitro and in vivo studies identified various antitumor activities of plant-derived and synthetic CBs, although there are some studies in which CBs might also promote tumor progression. The relevant data are summarized in the following chapters and the kinetic data for individual CBs are summarized in Table 1 .
For the treatment and recurrence prevention of ER-positive breast cancer, a long-term treatment with the selective estrogen receptor modulator (SERM) tamoxifen is now standard. Tamoxifen effectively blocks estrogen-related growth of cancer cells and increases the disease-free and overall survival in patients with ER-positive breast cancer. It is still the therapy of choice for the treatment of ER-positive breast cancer in premenopausal women [65,78]. Studies involving cancer cell lines showed that tamoxifen, a hydroxylated, biologically active metabolite, and several other newer SERMs, act as inverse agonists for CB1-R and CB2-R with considerable affinity (between nM and low µM concentrations). In ER-lacking cancer cells, tamoxifen modulates adenylate cyclase activity and causes an increase in the intracellular cAMP by modulating CB-R activity . In ER-negative breast cancer cell lines, tamoxifen also increases intracellular calcium levels via CB2-R activation .
In addition to CB2-R and CB1-R, alternative CB-Rs are also of interest for breast cancer therapy. High expression levels of GPR55 were found in human breast tumors and were related to worse prognoses. GPR55 was also highly expressed in MDA-MB-231 cells, a human breast cancer cell line with considerable metastatic potential (compared with less-metastatic MCF-7 cells) . The proliferative effects mediated by GPR55 are thought to be a result of ERK activation and downstream expression of proto-oncogene c-FOS .
In both ER-positive and ER-negative breast cancer cells, CBD activates the intrinsic apoptotic pathway by changing the mitochondrial membrane potential, activating the translocation of the BH3 interacting-domain death agonist to the mitochondria, and releasing cytochrome C from mitochondria .
AEA and 2-AG are produced on demand by cells and work to maintain homeostasis . They have a short half-life and are quickly degraded through transport protein reuptake and hydroxylation by either fatty acid amide hydrolase (FAAH) for AEA or monoacylglycerol lipase (MAGL) for 2-AG. Finally, arachidonic acid (AA) and ethanolamine, from AEA, and AA and glycerol, from 2-AG, are formed. Endocannabinoids are responsible for retrograde synaptic signaling in the central nervous system. They move across the synaptic cleft in order to bind and activate the presynaptic CB1-R, causing an inhibition of neurotransmitter release.
Cannabinoids (CBs) from Cannabis sativa provide relief for tumor-associated symptoms (including nausea, anorexia, and neuropathic pain) in the palliative treatment of cancer patients. Additionally, they may decelerate tumor progression in breast cancer patients. Indeed, the psychoactive delta-9-tetrahydrocannabinol (THC), non-psychoactive cannabidiol (CBD) and other CBs inhibited disease progression in breast cancer models. The effects of CBs on signaling pathways in cancer cells are conferred via G-protein coupled CB-receptors (CB-Rs), CB1-R and CB2-R, but also via other receptors, and in a receptor-independent way. THC is a partial agonist for CB1-R and CB2-R; CBD is an inverse agonist for both. In breast cancer, CB1-R expression is moderate, but CB2-R expression is high, which is related to tumor aggressiveness. CBs block cell cycle progression and cell growth and induce cancer cell apoptosis by inhibiting constitutive active pro-oncogenic signaling pathways, such as the extracellular-signal-regulated kinase pathway. They reduce angiogenesis and tumor metastasis in animal breast cancer models. CBs are not only active against estrogen receptor-positive, but also against estrogen-resistant breast cancer cells. In human epidermal growth factor receptor 2-positive and triple-negative breast cancer cells, blocking protein kinase B- and cyclooxygenase-2 signaling via CB2-R prevents tumor progression and metastasis. Furthermore, selective estrogen receptor modulators (SERMs), including tamoxifen, bind to CB-Rs; this process may contribute to the growth inhibitory effect of SERMs in cancer cells lacking the estrogen receptor. In summary, CBs are already administered to breast cancer patients at advanced stages of the disease, but they might also be effective at earlier stages to decelerate tumor progression.
As CBD is derived from CBDA by decaboxylation, it was investigated whether CBDA is also biologically active . Indeed, CBDA prevents migration of triple-negative MDA-MB-231 human breast cancer cells via CB2-R activation by modulating the expression and activity of COX-2 [59,60,61,62]. Furthermore, CBDA inhibits the growth and migration of breast cancer cells via the inhibition of cAMP-dependent protein kinase A via activation of the small GTPase, RhoA . It also causes a downregulation of the enhancer of breast cancer metastasis ID-1 .
Other CBs from C. sativa.
As described for CBD, antiproliferative actions of THC in tumor cells are caused by the activation of CB-Rs, which influence various signaling mechanisms ( Figure 2 ). Activation of CB2-R impairs cell cycle progression by downregulating cell division control 2 (Cdc2) and inducing cell cycle arrest at the G2/M phase. Furthermore, CB2-R causes an activation of a member of the activating protein 1 transcription family, transcription factor jun-D, preventing cell proliferation and inducing apoptosis [29,30].
Other CBs from C. sativa were also found to have anti-inflammatory and analgetic effects. Some of these CBs were found to improve the effects of inflammatory diseases in the gut and stimulate bone formation. These effects are conferred by the activation of CB-R and other receptors. Their concentration varies between different C. sativa strains but is generally as low as 2%. However, the concentrations of these CBs may reach significant levels in special cultivated strains. Additive or synergistic interactions between CBD, THC with minor phyto-CBs, or non-CBs, such as terpenes, in the extracts may increase the therapeutic efficiency of the extract for the treatment of inflammation and cancer [12,33].
Endocannabinoids work via specific G-protein coupled receptors (GPRs) CB-Rs (CB1-R and CB2-R). While AEA acts as a partial CB1-R agonist and is a weak CB2-R agonist, 2-AG is a strong CB1-R agonist. CB1-R and CB2-R belong to the seven-transmembrane-spanning receptor superfamily. The distinct tissue distribution of CB1-R and CB2-R allows a selective and cell-specific effect of receptor activation. CB1-R is highly expressed in brain areas related to cognitive functions, memory, anxiety, pain, sensory and visceral perception, motor coordination, and endocrine functions. Low expression levels are observed in the peripheral nervous system, testicles, heart, small intestine, prostate, uterus, bone marrow and vascular endothelium. CB1-R activations inhibit forskolin-stimulated adenylyl cyclase through activation of a pertussis toxin-sensitive G-protein, to inhibit N-, P-, and Q-type calcium channels, and activate inwardly rectifying potassium channels.
To target CB-R mediated pathways, compounds with different chemical structures were screened for CB-R receptor ligand activity. A number of these compounds were investigated in cell culture and animal tumor models to determine their antineoplastic effects. For relevant reviews, see references [37,38,39,40,41]. Their chemical structures are depicted in Figure 1 and their effects are discussed in the following chapters.
CB2-R is present at high levels in cells of the immune system. In glial cells, the spleen and tonsils, CB1-R levels are low. CB2-Rs are also present at a lower level in the heart, endothelium, bones, liver, and pancreas. Furthermore, a functionally relevant expression of CB2-Rs was also found in the brain . Intracellular CB2-R dependent signaling pathways include Gi/o-dependent inhibition of adenylyl cyclase, stimulation of mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and cyclooxygenase-2 (COX-2) signaling, and activation of de novo ceramide synthesis. Both CB-R types are highly expressed in a variety of cancerous tissues, and it is well established that CB2-R plays a crucial role in carcinogenesis and cancer progression. Therefore, CB2-R is now emerging as target for cancer treatment, although the exact role of CB2-R in cancer progression is still not completely elucidated .
Cultivation from different varieties of C. sativa produces two main varieties with distinct concentrations of CBs, and the discrimination from the THC/CBD ratio divides commercial cannabis strains into three principal chemotypes. Chemotype I flowers have the highest THC content (18–23%). Industrial C. sativa flowers (chemotype II and III flowers) contain less than 0.3% THC and CBD levels are 10–12% when calculated for dry weight . Since there are still systematic differences in reports on the CB content and the relative stability of CB levels from different laboratories, a better standardization of CB analysis is urgently required.
Chemical structures of cannabinoids. Phytocannabinoids —THC: Delta-9-tetrahydrocannabinol; THCA: Delta-9-tetrahydrocannabinolic acid; CBD: Cannabidiol; CBDA: Cannabidiolic acid; CBN: Cannabinol; CBG: Cannabigerol; CBC: Cannabichromene THCV: Tetrahydrocannabivarin. Endocannabinoids —AEA: Anandamide; 2-AG: 2-Arachidonoylglycerol; Met-F-AEA: 2-methyl-2’-F-anandamide; ACEA: Arachidonyl-2’-chloroethylamide. Synthetic cannabinoids —AM251: N -(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carbox amide; JW133: (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran-d5; WIN55,212-2: ( R )-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3- de ]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate; HU-331,CBDHQ: 3-Hydroxy-2-[(1 R )-6-isopropenyl-3-methyl-cyclohex-2-en-1-yl]-5-pentyl-1,4-benzoquinone; O-1663: 5-(1,1-Dimethylheptyl)-2-(4-phenylcyclohexyl)-1,3-benzenediol.
The acute toxicity of CBs is low in adults, but toxic effects occur mostly through THC. Inhaled doses of 2–3 mg THC and ingested doses of 5–20 mg THC can lead to impaired attention and memory, as well as in executive functioning, and conjunctivitis is a common symptom. Higher doses in adults and oral 5–300 mg in pediatric patients can cause more severe symptoms such as hypotension, panic, anxiety, delirium, respiratory depression and ataxia. Furthermore, chronic application of THC may lead to attention and memory deficits, as well as loss of the ability to process complex information. In children, neurological abnormalities, including lethargy and hyperkinesis, can be signs of severe toxicity. As THC crosses the placenta and accumulates to significant concentrations in breast milk, THC consumption by pregnant and breast-feeding women may harm unborn and newborn babies .
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