D-limonene: A Possible Role In Cancer Chemoprevention And Therapy
LPS与TLR-4/CD-14受体复合物的相互作用通常会激活巨噬细胞介导的先天免疫应答。TLR的胞外结构域与LPS结合，而胞内结构域激活一个包含多个蛋白的通路，序列精确:MyD88和MAL, IRAK1和IRAK4, TRAF6和TAK1。TAK1具有双重作用，激活了两种不同的通路:MAPK信号通路和IkB激酶。前者具有有丝分裂作用，使免疫细胞增殖;后者允许转录因子NF-kB向细胞核迁移。与DNA NF-kB交互修改细胞的基因表达促进多个基因的转录等炎症介质的编纂cox - 2,进气阀打开,il - 1、il - 6和TNFα。
使用d -柠檬烯处理的巨噬细胞，尽管暴露于LPS中，但与对照组相比，其炎症介质的浓度要低得多，而且它们的活性没有改变。这些蛋白的抑制与d -柠檬烯浓度呈线性关系。NF-kB信号的抑制似乎在这一现象中起着重要的作用，然而，迄今为止，d -柠檬烯影响免疫细胞中其他转录因子的活性是不可能排除的。
第二个可能也是最重要的活性和d -柠檬烯及其代谢物是它们对几种癌细胞系的细胞周期和生存能力的影响。研究表明，d -柠檬烯及其代谢产物具有下调Bcl-2表达的作用，Bcl-2是细胞凋亡线粒体通路的重要调控因子，可阻止BAX蛋白介导的凋亡孔的形成。Bcl-2常在癌细胞中过度表达。Bcl-2的下调和BAX的当代上调导致细胞质中细胞色素c的释放，从而激活caspase-9和caspase-3调控的凋亡机制。这一系列事件导致细胞凋亡。
最近的数据报告了另一种可能的机制，允许d -柠檬烯诱导线粒体死亡途径。据报道，该化合物可以下调PI3K/Akt/mTOR通路(通常在癌症中过度激活，允许增殖并防止凋亡)，从而导致BAD (Bcl-2相关死亡启动子)的激活，这是一种通过结合蛋白促进凋亡并阻断抗凋亡蛋白的蛋白。d -柠檬烯对PI3K/Akt/mTOR通路的抑制与剂量有关。这种作用机制已被报道在不同类型的癌症中发生，特别是在白血病细胞、结肠癌细胞甚至乳腺癌细胞上。
RAS蛋白的加工需要在碱基末端的第四个氨基酸位置的半胱氨酸残基(序列CaaX，其中C是半胱氨酸，a是脂族氨基酸，X是可变氨基酸)处进行最后的farnesylation或geranylgeranylation，以获得功能蛋白。这种半胱氨酸残基的突变阻断了RAS活性。farnesylation或geranylgeranyltransferase 1 (GGT1)由两种酶完成。两个酶都共享一个叫做FNTα功能单元,这种蛋白质裂解caspase-3在细胞凋亡从而表明某种信号可以通过间接诱导细胞凋亡抑制蛋白参与细胞的prenylation生存和增殖。
此外，d -柠檬烯和果酸以不同的方式影响甲戊酸酯通路，从而干扰法尼西聚丙烯和香叶香叶聚丙烯的产生，进而影响到丙烯酰化蛋白的产生(不仅仅是RAS)。事实上，最近的数据表明，紫苏酸和d -柠檬烯具有调节基因表达、翻译效率和翻译后处理3-羟基-3-甲基戊二酰辅酶A还原酶(HMG-CoA还原酶)的能力。有证据表明暴露于紫苏酸和d -柠檬烯的细胞中HMG-CoA还原酶的质量和活性降低。
D-limonene (1-methyl-4-(1-methylethenyl)cyclohexane) (molecular formula: C10H16 ) is a common cyclic terpene in nature. It is contained in the oil of several citrus fruits such as oranges, mandarins, grapefruit and lemons, from which it takes its name.
D-limonene and its metabolites have two different and clearly established effects on cell lines:
* pro-apoptotic on cancer cells
The anti-inflammatory activity has been studied on macrophages exposed to LPS (Lipopolysaccharide, the endotoxin present on Gram-negative bacteria outer cell membrane), a molecule which triggers a powerful immune response.
The interaction of LPS with the TLR-4/CD-14 receptor complex generally activate the innate immune response mediated by macrophages. The extracellular domains of the TLR binds the LPS while the intracellular domains activate a pathway involving several proteins in precise sequence: MyD88 and MAL, IRAK1 and IRAK4, TRAF6, TAK1. TAK1 has a double effect, activating two different pathways: the MAPK signaling pathway and the IkB kinase. The former has a mitotic effect, allowing immune cells proliferation; the latter permits the migration of the transcription factor NF-kB into the nucleus. Interacting with DNA NF-kB modifies the cell profile of gene expression promoting the transcription of several genes codifying for inflammatory mediators such as COX-2, iNOS, IL-1, IL-6 and TNFα.
Macrophages treated with D-limonene, despite being exposed to LPS, produce inflammatory mediators in much lower concentrations compared to controls while their viability is not modified. The inhibition of such proteins shows to be linearly connected with the concentration of D-limonene. The inhibition of the NF-kB signaling seems to have an important role in this phenomenon however, to date, it is impossible to exclude that D-limonene affects the activity of other transcription factors in immunity cells.
The second and maybe most important activity and D-limonene and its metabolites is their effect on cell cycle and viability in several cancer cell lines. It has been demonstrated that D-limonene and its metabolites have the effect of down regulating the expression of Bcl-2, an important regulator of the mitochondrial pathway of apoptosis which prevent the formation of the apoptotic pores mediated by the BAX protein. Bcl-2 is often overexpressed in cancer cells. The down regulation of Bcl-2 and the contemporary up regulation of BAX lead to the release of cytochrome c in the cytosol thus activating the apoptotic mechanism regulated by caspase-9 and caspase-3. This series of events results in apoptosis.
Recent data report another possible mechanism which permits D-limonene to induce the mitochondrial death pathway. The compound is reported to down regulate the PI3K/Akt/mTOR pathway (often over activated in cancer, allowing proliferation and preventing apoptosis) thus leading to the activation of BAD (Bcl-2 Associated Death promoter), a protein that promotes apoptosis and blocks anti apoptotic proteins by binding them. The inhibition of the PI3K/Akt/mTOR pathway by D-limonene is reported to be dose dependent. This mechanism of action has been reported to occur in different types of cancer, in particular on leukemia cell, on colon cancer cells and even on breast cancer cells.
More data is needed to assess D-limonene effects and possible clinical benefits on cancer patients.
The above reported phenomena are mainly due to the effects of D-limonene and related compounds on the activation of the RAS signaling network.
The processing of RAS protein requires a final farnesylation or geranylgeranylation at a cysteine residue at the fourth amino acid position from the COOH-terminal (sequence CaaX, in which C is cysteine, a is an aliphatic amino acid and X is variable amino acid) to obtain a functional protein. Mutations of that cysteine residue block RAS activity. Such reaction of farnesylation or geranylgeranylation is done by two enzymes: farnesyltransferase (FT) and geranylgeranyltransferase 1 (GGT1). Both enzymes share a functional subunit called FNTα, such protein is cleaved by caspase-3 during apoptosis thus suggesting that some kind of signal may induce apoptosis by indirectly inhibiting the prenylation of proteins involved in cell survival and proliferation.
D-limonene and perillic acid have been reported to be inhibitors of FT and GGT1 thus preventing the prenylation and subsequent activation of RAS and its related signaling cascades. A research group at St. Jude Children's Research Hospital in Memphis, USA, is working about the hypothesis that such compounds occupy an hydrophobic pocket of the prenyltransferases enzymes consequently blocking their activity. In this way these chemicals interfere with the activation of RAS signaling.
Moreover D-limonene and perillic acid are reported to affect the mevalonate pathway in different ways, hence interfering with the production of farnesyl-PP and geranylgeranyl-PP and consequently, with the production of prenylated proteins (not only RAS). In fact recent data proposed that perillic acid and also D-limonene have the ability to modulate gene expression, translational efficiency and also post-translational processing of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). There is evidence of a reduction of HMG-CoA reductase mass and activity in cells exposed to perillic acid and D-limonene.
The prevailing activity of such compounds seems to be the inhibition of FT and GGT1, thus interfering with the RAS signaling network, which affects not only the cell survival and proliferation but also interacts with the PI3K/Akt pathway and consequently with the NF-kB signaling.
The reduction of RAS prenylation could be an unifying explanation for both the anti-inflammatory and anticancer effects of terpenes contained in citrus fruits.
D-limonene induces apoptosis of gastric cancer cells
To investigate the apoptosis effect induced by D-limonene on BGC-823 gastric cancer cells. The expression of p53, bc1-2 in BGC-823 cells and qualitative, quantitative index of cell apoptosis were detected with MTT, electron microscopy, flow cytometry and immunohistochemical method. D-limonene could induce the formation of apoptotic bodies in a dose- and time-dependent manner. The expression of bcl-2 protein decreased and p53 protein increased in BGC-823 cells treated with D-limonene, compared with the control cells. D-limonene exerts its cytotoxic effect on BGC-823 gastric cancer cells by inducing apoptosis.
D-limonene induces apoptosis of gastric cancer cells | Request PDF. Available from: https://www.researchgate.net/publication/10610618_D-limonene_induces_apoptosis_of_gastric_cancer_cells [accessed Oct 10 2018].
As research continues and cannabis laws become more relaxed, it’s exciting to entertain the idea that more cannabis-derived medicines could replace potentially dangerous and addictive drugs that our current medical paradigm is so dependent upon.
According to The Leaf Online, a 2007 study from the University of Jordan set out to scientifically explore myrcene’s use as a folk remedy for diabetes. The researchers found that myrcene, hand in hand with another terpene named thujone, mitigated the effects of diabetes in a pilot study conducted on mice.
To reap the benefits of myrcene, it is advisable to select cannabis strains with profile high in the terpene. Lemongrass also holds a large amount of myrcene, which is believed to contribute to some of the herb’s therapeutic properties.