AB - Macrolide-pipecolate natural products, such as rapamycin (1) and FK-506 (2), are renowned modulators of FK506-binding proteins (FKBPs). The nocardiopsins, from Nocardiopsis sp. CMB-M0232, are the newest members of this structural class. Here, the biosynthetic pathway for nocardiopsins A-D (4-7) is revealed by cloning, sequencing, and bioinformatic analyses of the nsn gene cluster. In vitro evaluation of recombinant NsnL revealed that this lysine cyclodeaminase catalyzes the conversion of L-lysine into the L-pipecolic acid incorporated into 4 and 5. Bioinformatic analyses supported the conjecture that a linear nocardiopsin precursor is equipped with the hydroxy group required for macrolide closure in a previously unobserved manner by employing a P450 epoxidase (NsnF) and limonene epoxide hydrolase homologue (NsnG). The nsn cluster also encodes candidates for tetrahydrofuran group biosynthesis. The nocardiopsin pathway provides opportunities for engineering of FKBP-binding metabolites and for probing new enzymology in nature's polyketide tailoring arsenal. Expanding nature's macrolide-pipecolate biosynthetic repertoire: Cloning, sequencing, and analysis of the nocardiopsin biosynthetic pathway revealed that Nocardiopsis sp. CMB-M0232 employs a new strategy, featuring both a P450 epoxidase and an epoxide hydrolase, to transform the alkene group of a linear polyketide precursor into the hydroxy group required for macrolactonization.
AB - LH triggers the biosynthesis of androgens in the theca-interstitial (T-I) cells of ovary through the activation of a cAMP-dependent pathway. We have previously shown that LH/human chorionic gonadotropin (hCG) activates mammalian target of rapamycin complex 1 (mTORC1) signaling network, leading to cell proliferation. In the present study, we provide evidence that the LH/hCG-mediated activation of the mTORC1 signaling cascade is involved in the regulation of steroidogenic enzymes in androgen biosynthesis. Treatment with LH/hCG increased the expression of downstream targets of mTORC1, ribosomal protein S6 kinase 1, and eukaryotic initiation factor 4E as well as steroidogenic enzymes. LH/hCG-mediated stimulation of the steroidogenic enzyme mRNA was blocked by the mTORC1 inhibitor, rapamycin. This inhibitory effect was selective because rapamycin failed to block hCG-mediated increase in the expression of Star mRNA levels. Furthermore, pharmacological targeting of mTORC1 with rapamycin also blocked LH/hCG-or forskolin-induced expression of cAMP response element-binding protein (CREB) and steroidogenic enzymes (P450 side-chain cleavage enzyme, 3β-hydroxysteroid dehydrogenase type 1, and 17α-hydroxylase/17,20 lyase) but produced no effect on steroidogenic acute regulatory protein levels. These results were further confirmed by demonstrating that the knockdown of mTOR using small interfering RNA selectively abrogated the LH/hCG-induced increase in steroidogenic enzyme expression, without affecting steroidogenic acute regulatory protein expression. LH/hCG-stimulated androgen production was also blocked by rapamycin. Furthermore, the pharmacological inhibition of mTORC1 or ribosomal protein S6 kinase 1 signaling prevented the LH/hCG-induced phosphorylation of CREB. Chromatin immunoprecipitation assays revealed the association of CREB with the proximal promoter of the Cyp17a1 gene in response to hCG, and this association was reduced by rapamycin treatment. Taken together, our findings show for the first time that LH/hCG-mediated activation of androgen biosynthesis is regulated by the mTORC1 signaling pathway in T-I cells.
One of the important aspects of an effective anti-tumor drug is its ability to inhibit proliferation of cancer cells. Cancer cells proliferate rapidly in uncontrolled manner. Also, these cells escape death mechanism which a normal cell undergoes like apoptosis. Apoptosis is a kind of programmed cell death (PCD) mechanism which involves activation of caspase dependent and independent pathways . Cannabinoids have been proved to be anti-proliferative and apoptotic drugs. This section comprises of the detailed role of cannabinoids in modulation of tumor proliferation, cell cycle and apoptosis in various cancer types.
Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.
Id-1 (Inhibitor of differentiation/DNA binding) is a member of the helix-loop-helix protein family expressed in actively proliferating cells. It regulates gene transcription by heterodimerization with the basic helix-loop-helix transcription factors and therefore inhibits them from DNA binding and transactivation of their target genes. Early studies showed that Id-1 functions mainly as a regulator in cellular differentiation of the muscle cells. The oncogenic role of Id-1 was revealed recently by the finding that Id-1 expression was able to induce cancer cell growth and promote cell survival. In addition, Id-1 protein was frequently overexpressed in over 20 types of cancer, supporting its role in the tumorigenesis of a wide range of tissues. However, the fact that Id-1 was able to activate multiple pathways involved in tumor progression suggests that Id-1 may in addition function in promotion of tumor development. For example, overexpression of Id-1 was found to induce expression of MT1-MMP protein, leading to invasion of breast cancer cells. A close association between Id-1 expression and angiogenesis has also been demonstrated recently in both normal and cancer cells. Accordingly, in prostate cancer cells, expression of Id-1 was able to activate EGF-R and nuclear factor-kappaB activities and resulted in progression to androgen independence. In addition, in both nasopharyngeal carcinoma and prostate cancer cells, Id-1 expression was found to protect the cells from chemotherapeutic drug-induced apoptosis through regulation of the Raf-1/MAPK and JNK pathways. This review will discuss recent evidence supporting the role of Id-1 in tumor progression and the mechanisms involved.
Cancer Cell. 2003 Jun;3(6):525-30.
Id proteins in cell growth and tumorigenesis.
Sikder HA1, Devlin MK, Dunlap S, Ryu B, Alani RM.
Since the gene encoding Id1 was cloned in 1990, Id proteins have been implicated in regulating a variety of cellular processes, including cellular growth, senescence, differentiation, apoptosis, angiogenesis, and neoplastic transformation. The development of knockout and transgenic animal models for many members of the Id gene family has been particularly useful in sorting out the biologic relevance of these genes and their expression during normal development, malignant transformation, and tumor progression. Here we review the current understanding of Id gene function, the biologic consequences of Id gene expression, and the implications for Id gene regulation of cell growth and tumorigenesis.
Cellular differentiation programs are tightly controlled through the coordinated regulation of gene expression. Basic helix-loop-helix (bHLH) transcription factors regulate the differentiation programs of multiple cell lineages (reviewed in Norton, 2000). These proteins share a common sequence motif of a stretch of basic amino acids responsible for site-specific DNA binding adjacent to a helix-loop-helix dimerization domain. The Id family of helix-loop-helix proteins does not possess a basic DNA binding domain and functions as a dominant-negative regulator of basic HLH proteins through the formation of inactive heterodimers with intact bHLH transcription factors (Figure 1). The Id family of proteins (comprised of 4 members designated Id1–Id4) has been demonstrated to bind the ubiquitously expressed bHLH E-proteins or cell lineage-restricted bHLH transcription factors, leading to inhibition of lineage-specific gene expression and differentiation (Norton et al., 1998). Hence, the name Id refers to both inhibition of differentiation and inhibition of DNA binding. Transcriptional inhibition by Id proteins is mediated via inhibition of DNA binding of bHLH or other activator proteins at E boxes (CANNTG), N boxes (CACNAG), or Ets sites (GGAA/T) present in the promoter regions of regulated genes (reviewed in Zebedee and Hara, 2001). Since cellular differentiation programs are frequently altered during the development of neoplastic disease, it is not surprising that Id proteins would play a role in this process. Indeed, a clue to the potential role of Id genes in tumorigenesis came with the observation that, in general, high Id expression levels are found in proliferative, undifferentiated cells—a feature which is characteristic of tumor cells (Israel et al., 1999). Over the past several years, the particular mechanisms underlying the effects of Id genes on cell growth and differentiation have been investigated. Here we review data supporting the critical role of Id gene regulation in the development of normal cellular differentiation programs. We also review mechanisms of Id gene regulation of cellular growth controls and the cell cycle machinery and evaluate the contribution of dysregulated Id gene expression to the process of tumorigenesis.
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Kimura N, Tokunaga C, Dalal S, et al: Apossible linkage between AMP-activated protein kinase (AMPK) andmammalian target of rapamycin (mTOR) signalling pathway. GenesCells. 8:65–79. 2003. : :
Ramer’s group at the Unviersity of Rostock in Germany has done excellent work on CBD use in lung cancer with a 2012 published in FASEB. They found early-onset upregulation (four-fold) of ICAM-1 (intercellular adhesion molecule-1) via cannabinoid receptors in CDB treated lung cancer cells. Later, 48 hrs on, they found upregulation of the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) accounting for the loss of invasiveness of the lung cancer cells. In-vivo experiments injecting lung cancer cells (A549, H358, and H460) into mice then treated with CBD showed 2-3 fold increase in ICAM-1 and TIMP-1 protein which decreased cancer cell invasiveness. Upon microscopic inspection, the number of lung metastatic lesions had been reduced in half in the CBD treated mice. In yet another study, Ramers group also a new mechanism, the downregulation of the plasminogen activator inhibitor PAI-1, a protein involved in tumor invasiveness.
Kalender A, Selvaraj A, Kim SY, et al:Metformin, independent of AMPK, inhibits mTORC1 in a ragGTPase-dependent manner. Cell Metab. 11:390–401. 2010. : :
Dr. Massi’s medical literature review on the anticancer activity of cannabinoids again supports the “Whack-A-Mole” concept of cancer inhibition, with differing pathways and mechanisms found depending on the cancer model studied.(8) Dr. Massi’s review covers 5 different cancer types, Breast cancer, Glioma,Leukemia, thyroid cancer, colon cancer noting the type of cannabinoid receptor involvement, production of ROS (reactive oxygen), molecular cell signalling, and presence or absence of autophagy and apoptosis, (See for this information) (8).
In summary, we demonstrated that COH-SR4 suppressedadipogenesis in 3T3-L1 cells through indirect activation of AMPKand downstream modulation of the mTORC1 signaling pathway, whichblocked important regulators involved in protein synthesis, cellcycle progression, and expression of key transcription factors andtheir target adipogenic genes involved in lipid synthesis. Inaddition to exhibiting potent anticancer properties, COH-SR4 is apotential therapeutic candidate for the treatment and prevention ofobesity and related metabolic disorders. We are currently assessingthe pharmacological effects of COH-SR4 in diet-induced obese (DIO)mice as well as type 2 diabetic db/db mice.