The mechanistic target of rapamycin (mTOR) coordinates eukaryotic cell growth and metabolism with environmental inputs, including nutrients and growth factors. Extensive research over the past two decades has established a central role for mTOR in regulating many fundamental cell processes, from protein synthesis to autophagy, and deregulated mTOR signaling is implicated in the progression of cancer and diabetes, as well as the aging process. Here, we review recent advances in our understanding of mTOR function, regulation, and importance in mammalian physiology. We also highlight how the mTOR signaling network contributes to human disease and discuss the current and future prospects for therapeutically targeting mTOR in the clinic.

mTOR Signaling in Growth, Metabolism, and Disease

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that are involved in the degradation of various proteins in the extracellular matrix (ECM). Typically, MMPs have a propeptide sequence, a catalytic metalloproteinase domain with catalytic zinc, a hinge region or linker peptide, and a hemopexin domain. MMPs are commonly classified on the basis of their substrates and the organization of their structural domains into collagenases, gelatinases, stromelysins, matrilysins, membrane-type (MT)-MMPs, and other MMPs. MMPs are secreted by many cells including fibroblasts, vascular smooth muscle (VSM), and leukocytes. MMPs are regulated at the level of mRNA expression and by activation of their latent zymogen form. MMPs are often secreted as inactive pro-MMP form which is cleaved to the active form by various proteinases including other MMPs. MMPs cause degradation of ECM proteins such as collagen and elastin, but could influence endothelial cell function as well as VSM cell migration, proliferation, Ca2+ signaling, and contraction. MMPs play a role in tissue remodeling during various physiological processes such as angiogenesis, embryogenesis, morphogenesis, and wound repair, as well as in pathological conditions such as myocardial infarction, fibrotic disorders, osteoarthritis, and cancer. Increases in specific MMPs could play a role in arterial remodeling, aneurysm formation, venous dilation, and lower extremity venous disorders. MMPs also play a major role in leukocyte infiltration and tissue inflammation. MMPs have been detected in cancer, and elevated MMP levels have been associated with tumor progression and invasiveness. MMPs can be regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs), and the MMP/TIMP ratio often determines the extent of ECM protein degradation and tissue remodeling. MMPs have been proposed as biomarkers for numerous pathological conditions and are being examined as potential therapeutic targets in various cardiovascular and musculoskeletal disorders as well as cancer.

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Biochemical and Biological Attributes of Matrix Metalloproteinases.

mTOR complex 2 (mTORC2) contains the mammalian target of rapamycin (mTOR) kinase and the Rictor regulatory protein and phosphorylates Akt. Whether this function of mTORC2 is critical for cancer progression is unknown. Here, we show that transformed human prostate epithelial cells lacking PTEN require mTORC2 to form tumors when injected into nude mice. Furthermore, we find that Rictor is a haploinsufficient gene and that deleting one copy protects Pten heterozygous mice from prostate cancer. Finally, we show that the development of prostate cancer caused by Pten deletion specifically in prostate epithelium requires mTORC2, but that for normal prostate epithelial cells, mTORC2 activity is nonessential. The selective requirement for mTORC2 in tumor development suggests that mTORC2 inhibitors may be of substantial clinical utility.

mTOR Complex 2 Is Required for the Development of Prostate Cancer Induced by Pten Loss in Mice

Optical techniques using developed laser and optical devices have made a profound impact on modern medicine, with "biomedical optics" becoming an emerging field. Sophisticated technologies have been developed in cancer nanomedicine, such as photothermal therapy and photodynamic therapy, among others. However, single-mode phototherapy cannot completely treat persistent tumors, with the challenges of relapse or metastasis remaining; therefore, combinatorial strategies are being developed. In this review, the role of light in cancer therapy and the challenges of phototherapy are discussed. The development of combinatorial strategies with other therapeutic methods, including chemotherapy, immunotherapy, gene therapy, and radiotherapy, is presented and future directions are further discussed. This review aims to highlight the significance of light in cancer therapy and discuss the combinatorial strategies that show promise in addressing the challenges of phototherapy.

Emerging combination strategies with phototherapy in cancer nanomedicine

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade various proteins in the extracellular matrix (ECM). Typically, MMPs have a propeptide sequence, a catalytic metalloproteinase domain with catalytic zinc, a hinge region or linker peptide, and a hemopexin domain. MMPs are commonly classified on the basis of their substrates and the organization of their structural domains into collagenases, gelatinases, stromelysins, matrilysins, membrane-type (MT)-MMPs, and other MMPs. MMPs are secreted by many cells including fibroblasts, vascular smooth muscle (VSM), and leukocytes. MMPs are regulated at the level of mRNA expression and by activation through removal of the propeptide domain from their latent zymogen form. MMPs are often secreted in an inactive proMMP form, which is cleaved to the active form by various proteinases including other MMPs. MMPs degrade various protein substrates in ECM including collagen and elastin. MMPs could also influence endothelial cell function as well as VSM cell migration, proliferation, Ca2+ signaling, and contraction. MMPs play a role in vascular tissue remodeling during various biological processes such as angiogenesis, embryogenesis, morphogenesis, and wound repair. Alterations in specific MMPs could influence arterial remodeling and lead to various pathological disorders such as hypertension, preeclampsia, atherosclerosis, aneurysm formation, as well as excessive venous dilation and lower extremity venous disease. MMPs are often regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs), and the MMP/TIMP ratio often determines the extent of ECM protein degradation and tissue remodeling. MMPs may serve as biomarkers and potential therapeutic targets for certain vascular disorders.

Matrix Metalloproteinases, Vascular Remodeling, and Vascular Disease

Cell adhesion molecules (CAMs) are cell surface glycoproteins that facilitate cell-cell contacts and adhesion with the extracellular matrix (ECM). Cellular adhesion is affected by various disease conditions, such as diabetes mellitus (DM) and inflammation. Photobiomodulation (PBM) stimulates biological processes and expression of these cellular molecules. The aim of this experimental work was to demonstrate the role of PBM at 830nm on CAMs in diabetic wounded fibroblast cells. Isolated human skin fibroblast cells were used. Normal (N-) and diabetic wounded (DW-) cells were irradiated with a continuous wave diode laser at 830nm with an energy density of 5J/cm(2). Real time reverse transcriptase polymerase chain reaction (RT-PCR) was used to determine the relative gene expression of 39 CAMs 48h post-irradiation. Normalized expression levels from irradiated cells were calculated relative to non-irradiated control cells according to the 2^(-ΔΔCt) method. Thirty-one genes were significantly regulated in N-cells (28 were genes up-regulated and three genes down-regulated), and 22 genes in DW-cells (five genes were up-regulated and 17 genes down-regulated). PBM induced a stimulatory effect on various CAMs namely cadherins, integrins, selectins and immunoglobulins, and hence may be used as a complementary therapy in advancing treatment of non-healing diabetic ulcers. The regulation of CAMs as well as evaluating the role of PBM on the molecular effects of these genes may expand knowledge and prompt further research into the cellular mechanisms in diabetic wound healing that may lead to valuable clinical outcomes.

The role of photobiomodulation on gene expression of cell adhesion molecules in diabetic wounded fibroblasts in vitro

The integration of several cellular responses initiates the process of wound healing. Matrix Metalloproteinases (MMPs) play an integral role in wound healing. Their main function is degradation, by removal of damaged extracellular matrix (ECM) during the inflammatory phase, breakdown of the capillary basement membrane for angiogenesis and cell migration during the proliferation phase, and contraction and remodelling of tissue in the remodelling phase. For effective healing to occur, all wounds require a certain amount of these enzymes, which on the contrary could be very damaging at high concentrations causing excessive degradation and impaired wound healing. The imbalance in MMPs may increase the chronicity of a wound, a familiar problem seen in diabetic patients. The association of diabetes with impaired wound healing and other vascular complications is a serious public health issue. These may eventually lead to chronic foot ulcers and amputation. Low intensity laser irradiation (LILI) or photobiomodulation (PBM) is known to stimulate several wound healing processes; however, its role in matrix proteins and diabetic wound healing has not been fully investigated. This review focuses on the role of MMPs in diabetic wound healing and their interaction in PBM.

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The Role of Matrix Metalloproteinases in Diabetic Wound Healing in relation to Photobiomodulation

Background: Collagen type I (Col-I) is a major component of the extracellular matrix and is important in wound healing processes. Several studies have shown that low-intensity laser irradiation (LILI) biostimulates Col-I synthesis both in vitro and in vivo. This study aimed to determine if LILI affects collagen production and related cellular responses in an in vitro diabetic wounded fibroblast model. Materials and Methods: This study was performed on isolated human skin fibroblasts. Different cell models (normal and diabetic wounded) were used. Cells were irradiated with 5 J/cm2 at a wavelength of 660 nm and incubated for 48 or 72 h. Nonirradiated cells (0 J/cm2) were used as controls. Cellular viability (Trypan blue exclusion test), morphology (bright-field microscopy), proliferation [VisionBlue™ quick cell proliferation assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay], and Col-I (enzyme-linked immunoabsorbent assay) were assessed. Results: Diabetic wounded cells ir...

Collagen production in diabetic wounded fibroblasts in response to low-intensity laser irradiation at 660 nm.

Photobiomodulation has been found to increase gene expression and release of various growth factors and cytokines involved in wound healing. Photobiomodulation has been used to treat a wide variety of disorders, and has been found to be beneficial in the treatment of chronic wounds, however the exact underlying mechanism is not well understood. This study aimed to profile 84genes in response to irradiation at 660nm. WS1 human skin fibroblasts were used in gene expression profiling studies in response to irradiation with a 660nm diode laser at a fluence of 5J/cm(2) and power density of 11mW/cm(2). Forty-eight hours post-irradiation, 1μg RNA was reverse transcribed and used in real-time qualitative polymerase chain reaction (qPCR). Genes involved in the extracellular matrix and cell adhesion, inflammatory cytokines and chemokines, growth factors and signal transduction were evaluated. A total of 76genes were regulated by laser irradiation, 43genes were up-regulated while 33genes were down-regulated. Irradiation of WS1 cells at 660nm modulates the expression of genes involved in collagen production, cellular adhesion, remodelling and spreading, the cytoskeleton, inflammatory cytokines and chemokines, growth factors and molecules involved in signal transduction.

Expression of genes in normal fibroblast cells (WS1) in response to irradiation at 660nm.

The current study evaluated the photobiomodulatory effect of visible red light on cell proliferation and viability in various fibroblast diabetic models in vitro, namely, unstressed normal (N) and stressed normal wounded (NW), diabetic wounded (DW), hypoxic wounded (HW) and diabetic hypoxic wounded (DHW). Cells were irradiated at a wavelength of 660 nm with a fluence of 5 J/cm2 (11.23 mW/cm2), which related to an irradiation time of 7 min and 25 s. Control cells were not irradiated (0 J/cm2). Cells were incubated for 48 h and cellular proliferation was determined by measuring 5-bromo-2′-deoxyuridine (BrdU) in the S-phase (flow cytometry), while viability was assessed by the Trypan blue exclusion test and Apoptox-glo triplex assay. In comparison with the respective controls, PBM increased viability in N- (P ≤ 0.001), HW- (P ≤ 0.01) and DHW-cells (P ≤ 0.05). HW-cells showed a significant progression in the S-phase (P ≤ 0.05). Also, there was a decrease in the G2M phase in HW- and DHW-cells (P ≤ 0.05 and P ≤ 0.05, respectively). This study concludes that hypoxic wounded and diabetic hypoxic wounded models responded positively to PBM, and PBM does not damage stressed cells but has a stimulatory effect on cell viability and proliferation to promote repair and wound healing. This suggests that the more stressed the cells are the better they responded to photobiomodulation (PBM).

Sandra M. Ayuk

Papers

The role of photobiomodulation on gene expression of cell adhesion molecules in diabetic wounded fibroblasts in vitro

The Role of Matrix Metalloproteinases in Diabetic Wound Healing in relation to Photobiomodulation

Collagen production in diabetic wounded fibroblasts in response to low-intensity laser irradiation at 660 nm.

Expression of genes in normal fibroblast cells (WS1) in response to irradiation at 660nm.

Effect of 660 nm visible red light on cell proliferation and viability in diabetic models in vitro under stressed conditions.