Cellular and Molecular Biology Focusing on the brighter side of Sevoflurane: Realizing true potential of an anesthetic agent as a regulator of cell signaling pathways and microRNAs in different cancers

: Reconceptualization of different anesthetics as anticancer agents has opened new horizons for a better and sharper analysis of true potential of Sevoflu rane as a promising and frontline candidate in the pipeline of anticancer agents. Sevoflurane mediated regulation of cell signaling pathways and non-coding RNAs has leveraged our understanding to another level. There have been remarkable advancements in unraveling mechanistic insights related to the ability of sevoflurane to modulate microRNAs in different cancers. Astonishingly, sevoflurane mediated regulation of miRNAs and long non-coding RNAs have been more comprehen sively addressed in ischemia-reperfusion injuries. However, researchers yet have to gather missing pieces of premium research-work to uncover mechanistic regulation of long non-coding RNAs by sevoflurane in various cancers. Sevoflurane modulated control of miRNAs have been reported in glioma, colorectal cancer, breast cancer and hepatocellular carcinoma. In this review we have attempted to summarize most recent cutting edge and high-impact experimental researches which have elucidated myriad of underlying mechanisms modulated by sevoflurane to inhibit cancer development and progression. Despite some of the amazing pharmacological properties of sevoflurane, it has been shown to possess darker side because of its involvement in positive regulation of metastasis. In accordance with this notion we have also summarized how sevoflurane enhanced migratory potential of different cancer cells in a separate section. Therefore, these aspects have to be tested in better designed experimental models to identify most relevant types of cancers which can be therapeutically targeted by sevoflurane.


Introduction
Based on the rapidly emerging evidence related to use of volatile anesthetics as modulators of deregulated cell signaling pathways in different cancers, interdisciplinary researchers have witnessed a sharp division of opinion between opponents and proponents. Excitingly, this difference of opinion has impelled researchers to drill down deep into the cellular and molecular biology for re-consideration of volatile anesthetics as anticancer agents. Dissemination of tumor cells during surgery has remained a major stumbling block and one of the major causes of post-operative metastasis. Wealth of information obtained through a critical analysis of the effects exerted by anesthetics at cellular levels has sparked an interest in unraveling the gene regulatory networks modulated by these anesthetics. In this review we have set spotlight on ability of sevoflurane to modulate oncogenic protein network and microRNAs in different cancers.

Regulation of cell signaling pathways by sevoflurane in different cancers
Deregulation of cell signaling pathways played a central role in cancer development and progression. In this section, we will summarize recent updates related to regulation of different oncogenic proteins and transduction cascades in different cancers by sevoflurane.
Sevoflurane inhibited migration of SCC-4 cells. Sevoflurane attenuated hypoxia-induced stimulation of VEGF in tongue squamous cell carcinoma cells by enhancement of DNA methylation at promoter region of VEGF (1).
Sevoflurane exerted inhibitory effects on platelets activation of patients undergoing lung cancer surgery. GPIIb/IIIa also known as αIIbβ3 and CD62P are present on surface of platelets and have been shown to centrally regulate lung metastasis of cancer cells (2). Levels of CD62P and GPIIb/IIIa were found to be considerably enhanced in platelets activated after surgery. However, sevoflurane significantly reduced the levels of CD62P and GPIIb/IIIa and remarkably counteracted plateletsinduced invasive potential of lung cancer cells (2). Overall these findings clearly suggested that sevoflurane suppressed platelets-mediated increase in invasive capacity of lung cancer cells.
Different clinical researches have shown that regional anesthesia improved post-operative outcomes and reduced the chances of infection by exerting immunostimulatory effects. Liver mononuclear cells (MNCs) include NK cells and natural killer T (NKT) cells which effectively inhibit liver metastasis and have been shown to play critical and instrumental role in inhibiting pulmonary metastases (3). Surgical stress has remained focus of discussion because it repressed cytotoxic activities of liver MNCs and simultaneously enhanced the growth of metastatic liver tumors (3). More importantly, liver MNC function was found to be more sensitive to surgical stress as compared to peripheral blood mononuclear cell function. Spinal block and sevoflurane combinatorially safeguarded the functionality of liver MNCs by maintenance of balance in the T helper 1 (Th1)/ Th2 in response to surgery and reduced metastasis after surgery (3).
Activated Rho proteins bind to and activate ROCK1 and ROCK2 which phosphorylate MYPT1 and MLC. Shown in figure 1. Sevoflurane markedly inhibited phosphorylation of MYPT1 (myosin phosphatase-targeting subunit 1), MLC (myosin light chain), ERK and AKT (4). Overall these findings provided clear proof that sevoflurane not only suppressed RhoA and Ras activities but also downstream signaling pathways centrally involved in uncontrolled proliferation and migration of cervical cancer cells.
Combinatorial targeting of ROCK1 and MEK/ERK pathway has been shown to enhance killing of cancer cells (5,6).
Sevoflurane dose-dependently reduced β-catenin levels and its downstream effectors in CML CD34 and K562 cells (7). Additionally, Wnt/β-catenin-induced transcriptional upregulation of MYC, CYCLIN D1 and BCL9 was also found to be remarkably reduced in K562 cells exposed to sevoflurane. Shown in figure. These findings indicated that sevoflurane inhibited Wnt/βcatenin pathway in CML cells (7).
Sevoflurane suppressed hypoxia-induced growth and metastasizing potential of lung cancer cells (8). Likewise, HIF-1α, XIAP and survivin were found to be considerably downregulated in lung cancer cells co-treated with sevoflurane and hypoxic conditions as compared to hypoxia treatment alone. Dimethyloxaloylglycine (DMOG), a HIF-1α agonist severely abolished the inhibitory effects exerted by sevoflurane on hypoxia-induced growth and metastasizing ability of lung cancer cells (8).

Regulation of microRNAs by Sevoflurane in different cancers
Non-coding RNAs have revolutionized the field of molecular oncology 10,11,12 . There has been an explosion in the cancer biology and non-coding RNAs have been shown to modulate wide-ranging oncogenic and tumor suppressor mRNAs 13,14 . In this section, we will discuss how sevoflurane modulated different microRNAs to regulate cancer development and progression.

Hepatocellular carcinoma
Phosphatase and tensin homolog (PTEN) is a tumor suppressor that classically inhibits the PI3K/AKT/ mTOR signaling pathway. Experimental models of PTEN loss have uncovered the molecular mechanisms which drive carcinogenesis. These insights have not only taught us about biological mechanisms but also enabled us to search for strategies to activate PTEN and inhibit PI3K/AKT pathway.
Mechanistically it was shown that sevoflurane enhanced PTEN expression and simultaneously reduced phosphorylated levels of p-PI3K and p-AKT in hepatocellular carcinoma cells (15). Sevoflurane also stimulated the expression of miR-29a in HCC cells. miR-29a directly targeted DNMT3a (DNA methyltransferase 3a) and inhibited PI3K/AKT pathway via activation of PTEN (15).

Glioma
Sevoflurane treatment inhibited migratory and invasive potential of glioma cells (16). Excitingly, miR-146b-5p was enhanced whereas MMP16 levels were found to be notably reduced in sevoflurane-exposed glioma cells. It has been experimentally verified that overexpression of MMP16 or miR-146b-5p knockdown caused reversal of sevoflurane-induced inhibition of migration and invasion of glioma cells (16). ROCK1 (Rho-associated, coiled-coil-containing protein kinase-1) is a serine/threonine kinase (17). ROCK1 plays integral role in enhancing migratory and invasive potential of glioma cells. miR-124-3p directly targeted ROCK1. It was shown that miR-124-3p quantitatively controlled the expression of ROCK1 in sevo-  tially through inhibition of LFA-1 (25).
Sevoflurane time-and dose-dependently upregulated HIF-1α, HIF-2α, VEGF and p-AKT in glioma stem cells (26). RNA interference strategy against HIFs considerably reduced the percentage of proliferating GSCs after exposure of sevoflurane. Similarly, pre-treatment with AKT inhibitor also interfered with sevofluranemediated upregulation of HIFs (26). Collectively these findings suggested that sevoflurane potentiated the proliferation of GSCs through HIFs driven pathway.
Calpains belong to the family of cysteine proteases that catalyze the proteolysis of a large number of specific substrates. Sevoflurane exposure concentration-dependently enhanced the activity of calpains and CD44 protein in A172 and U251 cells (27). CD44 Knockdown with siRNA abrogated sevoflurane-induced increase in calpain activity, migratory, invasive and colony-forming capacity of U251 cells. It was observed that inhalation of 4% sevoflurane induced significant increase in the tumor volume, migration and invasion in nude mice xenografted with glioblastoma U87 cells (27).

Concluding remarks
Series of research reports have started to highlight preventive/inhibitory role of different anesthetics against cancer. Sevoflurane has gradually gained attention because of its promising ability to inhibit cancer progression. However, despite the initial promising results obtained in cancer cell lines, sevoflurane has to pass touchstone tests in molecular oncology. There are visible knowledge gaps in our understanding about ability of sevoflurane to modulate oncogenic cell signaling pathways.
It has recently been revealed that sevoflurane and propofol differentially influenced miRNAs in circulating extracellular vesicles during colorectal cancer resection (28). Sevoflurane has been shown to modulate various sets of miRNAs in different cancers but these findings need additional support by exposure of tumor bearing mice to sevoflurane. flurane-exposed glioma cells (17).
Phosphorylated AKT levels were found to be drastically reduced in miR-637 overexpressing glioma cells. Sevoflurane stimulated the expression of miR-637 and exerted repressive effects on p-AKT levels (18).

Colorectal cancer
miR-34a has emerged as a versatile tumor suppressor miRNA having scientifically validated potential to target myriad of oncogenic mRNAs in different cancers. Sevoflurane effectively stimulated the expression of miR-34a in colorectal cancer cells 19 . However, these effects were reversed by pre-treatment with miR-34a inhibitors. ADAM10 (A Disintegrin and metalloproteinase domain-containing protein-10) was critical in various stages of colorectal cancer. ADAM10 was negatively regulated by miR-34a in colorectal cancer cells. ADAM10 overexpression interfered with miR-34a and sevoflurane-induced inhibitory effects on proliferation, migration and invasion abilities of CRC cells (19).
Notch signaling pathway played central role in progression of colorectal cancer (20). Therapeutic targeting of Notch pathway will certainly be helpful in improving drug sensitivity. miR-34a pleiotropically modulated myriad of genes (21). Therefore, ADAM10 inhibition and miR-34a re-expression will inhibit migratory and invasive potential of cancer cells.
Roundabout1 (Robo1) also played central role in progression of colorectal cancer. Sevoflurane potentiated miR-203 mediated targeting of Robo1 and also reduced phosphorylated levels of ERK in CRC cells (22). miR-203 inhibition markedly abolished the inhibitory effects of sevoflurane on phosphorylated ERK levels in CRC cells (22).
Overall these findings provided clear hints about potential role of sevoflurane in regulation of tumor suppressor miRNAs. Future studies will converge on identification of additional tumor suppressor and oncogenic miRNAs which can be effectively modulated by sevoflurane in different cancers.
Osteopontin (OPN) is a secreted phosphoprotein frequently overexpressed in many cancers. Sevoflurane was found to enhance migratory potential of non-small cell lung cancer cells (A549) and renal cell carcinoma (RCC4) 24 . Sevoflurane upregulated TGFβ1, TGFβRII and OPN but reduced nuclear SMAD3 in cisplatintreated RCC4 cells. However, surprisingly sevoflurane potentiated migratory ability of A549 cells by increment of nuclear SMAD3 levels (24).
LFA-1 (Leukocyte function-associated antigen-1) is an essential adhesion molecule on NK cells25. Sevoflurane and Isoflurane abrogated the conjugation of K562 cells with NK92-MI cells. Sevoflurane and Isoflurane attenuated tumor cytotoxicity of natural killer cells par-