A Multicenter Phase 2 Trial Evaluating the Efficacy and Safety of Preoperative Lenvatinib Therapy for Patients with Advanced Hepatocellular Carcinoma (LENS-HCC Trial).

Introduction: The phase III REFLECT trial demonstrated that lenvatinib was superior to sorafenib in terms of progression-free survival (PFS), time to progression, and objective response rate (ORR) for patients with unresectable hepatocellular carcinoma (HCC). This study assessed the efficacy and safety of preoperative lenvatinib therapy for patients with oncologically or technically unresectable HCC. Methods: In this multicenter single-arm phase II trial, patients with advanced HCC and factors suggestive of a poor prognosis (macroscopic vascular invasion, extrahepatic metastasis, or multinodular tumors) were enrolled. Patients with these factors, even with technically resectable HCC, were defined as oncologically unresectable because of the expected poor prognosis after surgery. After 8 weeks of lenvatinib therapy, the patients were assessed for resectability, and tumor resection was performed if the tumor was considered technically resectable. The primary endpoint was the surgical resection rate. The secondary endpoints were the macroscopic curative resection rate, overall survival (OS), ORR, PFS, and the change in the indocyanine green retention rate at 15 min as measured before and after lenvatinib therapy. The trial was registered with the Japan Registry of Clinical Trials (s031190057). Results: Between July 2019 and January 2021, 49 patients (42 oncologically unresectable patients and 7 technically unresectable patients) from 11 centers were enrolled. The ORR was 37.5% based on mRECIST and 12.5% based on RECIST version 1.1. Thirty-three patients underwent surgery (surgical resection rate: 67.3%) without perioperative mortality. The surgical resection rate was 76.2% for oncologically unresectable patients and 14.3% for technically unresectable patients. The 1-year OS rate and median PFS were 75.9% and 7.2 months, respectively, with a median follow-up period of 9.3 months. Conclusions: The relatively high surgical resection rate seen in this study suggests the safety and feasibility of lenvatinib therapy followed by surgical resection for patients with oncologically or technically unresectable HCC.

Protocol of the RACB study: a multicenter, single-arm, prospective study to evaluate the efficacy of resection of initially unresectable hepatocellular carcinoma with atezolizumab combined with bevacizumab.

BACKGROUND: Although the standard therapy for advanced-stage hepatocellular carcinoma (HCC) is systemic chemotherapy, the combination of atezolizumab and bevacizumab (atezo + bev) with a high objective response rate may lead to conversion to resection in patients with initially unresectable HCC. This study aims to evaluate the efficacy of atezo + bev in achieving conversion surgery and prolonged progression-free survival (PFS) for initially unresectable HCC. METHODS: The RACB study is a prospective, single-arm, multicenter, phase II trial evaluating the efficacy of combination therapy with atezo + bev for conversion surgery in patients with technically and/or oncologically unresectable HCC. The main eligibility criteria are as follows: (1) unresectable HCC without a history of systemic chemotherapy, (2) at least one target lesion based on RECIST ver. 1.1, and (3) a Child‒Pugh score of 5-6. The definition of unresectable tumors in this study includes macroscopic vascular invasion and/or extrahepatic metastasis and massive distribution of intrahepatic tumors. Patients will be treated with atezolizumab (1200 mg/body weight) and bevacizumab (15 mg/kg) every 3 weeks. If the patient is considered resectable on radiological assessment 12 weeks after initial chemotherapy, the patient will be treated with atezolizumab monotherapy 3 weeks after combination chemotherapy followed by surgery 3 weeks after atezolizumab monotherapy. If the patient is considered unresectable, the patient will continue with atezo + bev and undergo a radiological assessment every 9 weeks until resectable or until disease progression. The primary endpoint is PFS, and the secondary endpoints are the overall response rate, overall survival, resection rate, curative resection rate, on-protocol resection rate, and ICG retention rate at 15 min after atezo + bev therapy. The assessments of safety and quality of life during the treatment course will also be evaluated. The number of patients has been set at 50 based on the threshold and the expected PFS rate at 6 months after enrollment of 40% and 60%, respectively, with a one-sided alpha error of 0.05 and power of 0.80. The enrollment and follow-up periods will be 2 and 1.5 years, respectively. DISCUSSION: This study will elucidate the efficacy of conversion surgery with atezo + bev for initially unresectable HCC. In addition, the conversion rate, safety and quality of life during the treatment course will also be demonstrated. TRIAL REGISTRATION: This study is registered in the Japan Registry of Clinical Trials (jRCTs051210148, January 7, 2022).

Alteration in MARCKS phosphorylation and expression by methylmercury in SH-SY5Y cells and rat brain.

The molecular mechanisms mediating methylmercury (MeHg)-induced neurotoxicity are not completely understood. Because myristoylated alanine-rich C kinase substrate (MARCKS) plays an essential role in the differentiation and development of neuronal cells, we studied the alteration of MARCKS expression and phosphorylation in MeHg-induced neurotoxicity of neuroblastoma SH-SY5Y cells and in the rat brain. Exposure to MeHg induced a decrease in cell viability of SH-SY5Y cells, which was accompanied by a significant increase in phosphorylation and a reduction in MARCKS expression. Pretreatment of cells with a protein kinase C inhibitor or an extracellular Ca(2+) chelator suppressed MeHg-induced MARCKS phosphorylation. In MARCKS knock-down cells, MeHg-induced cell death was significantly augmented in comparison to control siRNA. In brain tissue from MeHg-treated rats, MARCKS phosphorylation was enhanced in the olfactory bulb in comparison to control rats. The present study may indicate that alteration in MARCKS expression or phosphorylation has consequences for MeHg-induced neurotoxicity.

MARCKS dephosphorylation is involved in bradykinin-induced neurite outgrowth in neuroblastoma SH-SY5Y cells.

Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-ß and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1 min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10 min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.

MARCKS regulates lamellipodia formation induced by IGF-I via association with PIP2 and beta-actin at membrane microdomains.

Myristoylated alanine-rich C kinase substrate (MARCKS) is considered to participate in formation of F-actin-based lamellipodia, which represents the first stage of neurite formation. However, the mechanism of how MARCKS is involved in lamellipodia formation is not precisely unknown. Using SH-SY5Y cells, we demonstrated here that MARCKS was translocated from cytosol to detergent-resistant membrane microdomains, known as lipid rafts, within 30 min after insulin-like growth factor-I (IGF-I) stimulation, which was accompanied by MARCKS dephosphorylation, beta-actin accumulation in lipid rafts, and lamellipodia formation. The protein kinase C inhibitor, Ro-31-8220, and Rho-kinase inhibitors, HA1077 and Y27632, themselves decreased basal phosphorylation levels of MARCKS and coincidently elicited translocation of MARCKS to lipid rafts. On the other hand, the phosphoinositide 3-kinase inhibitor, LY294002, abolished IGF-I-induced dephosphorylation, translocation of MARCKS to lipid rafts, and lamellipodia formation. Treatment of cells with neomycin, a PIP2-masking reagent, attenuated the translocation of MARCKS to lipid rafts and the lamellipodia formation induced by IGF-I, although dephosphorylation of MARCKS was not affected. Immunocytochemical and immunoprecipitation analysis indicated that IGF-I stimulation induced the translocation of MARCKS to lipid rafts in the edge of lamellipodia and formation of the complex with PIP2. Moreover, we demonstrated that knockdown of endogenous MARCKS resulted in significant attenuation of IGF-I-induced beta-actin accumulation in the lipid rafts and lamellipodia formation. These results suggest a novel role for MARCKS in lamellipodia formation induced by IGF-I via the translocation of MARCKS, association with PIP2, and accumulation of beta-actin in the membrane microdomains.

Linalyl acetate as a major ingredient of lavender essential oil relaxes the rabbit vascular smooth muscle through dephosphorylation of myosin light chain.

In a preliminary experiment, we found that lavender essential oil relaxes vascular smooth muscle. Thus, the present experiments were designed to investigate the relaxation mechanism of linalyl acetate as the major ingredient of lavender essential oil in rabbit carotid artery specimens. Linalyl acetate produced sustained and progressive relaxation during the contraction caused by phenylephrine. The relaxation effect of linalyl acetate at a concentration near the EC50 was partially but significantly attenuated by nitroarginine as an inhibitor of nitric oxide synthase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one as an inhibitor of guanylyl cyclase, or by the denudation of endothelial cells. In specimens without endothelium, the phenylephrine-induced contraction and phosphorylation of myosin light chain (MLC) were significantly attenuated after the pretreatment with linalyl acetate. The relaxation caused by linalyl acetate in the endothelium-denuded specimens was clearly inhibited by calyculin A as an inhibitor of MLC phosphatase, although not by ML-9 as an inhibitor of MLC kinase. Furthermore, suppression of the phenylephrine-induced contraction and MLC phosphorylation with linalyl acetate was canceled by the pretreatment with calyculin A. These results suggest that linalyl acetate relaxes the vascular smooth muscle through partially activation of nitric oxide/cyclic guanosine monophosphate pathway, and partially MLC dephosphorylation via activating MLC phosphatase.

Unphosphorylated MARCKS is involved in neurite initiation induced by insulin-like growth factor-I in SH-SY5Y cells.

Myristoylated alanine-rich C kinase substrate (MARCKS) has been suggested to be involved in various aspects of neuronal cell differentiation, including neurite outgrowth. However, the precise mechanisms by which MARCKS phosphorylation is regulated, and how MARCKS contributes to neurite outgrowth, are poorly understood. Here, we found that treatment of SH-SY5Y cells with insulin-like growth factor-I (IGF-I) induced a rapid and transient decrease in the level of phosphorylated MARCKS (P-MARCKS) to below the basal level. The decrease in P-MARCKS induced by IGF-I was blocked by pretreatment of cells with phosphoinositide 3-kinase (PI3K) inhibitors, LY294002 and wortmannin. A decrease in P-MARCKS was also observed in cells treated with a Rho-dependent kinase (ROCK) inhibitor, Y27632. Furthermore, IGF-I induced transient inactivation of RhoA, an upstream effector of ROCK. We showed that MARCKS was translocated to the membrane and colocalized with F-actin at the lamellipodia and the tips of neurites in the cells stimulated with IGF-I. Finally, overexpression of wild-type MARCKS or an unphosphorylatable mutant of MARCKS enhanced the number of neurite-bearing cells relative to vector-transfected cells. Taken together, these findings suggest that unphosphorylated MARCKS is involved in neurite initiation, and highlight the important role played by MARCKS in organization of the actin cytoskeleton.

MARCKS is a major PKC-dependent regulator of calmodulin targeting in smooth muscle.

Calmodulin (CaM) is a ubiquitous transducer of intracellular Ca(2+) signals and plays a key role in the regulation of the function of all cells. The interaction of CaM with a specific target is determined not only by the Ca(2+)-dependent affinity of calmodulin but also by the proximity to that target in the cellular environment. Although a few reports of stimulus-dependent nuclear targeting of CaM have appeared, the mechanisms by which CaM is targeted to non-nuclear sites are less clear. Here, we investigate the hypothesis that MARCKS is a regulator of the spatial distribution of CaM within the cytoplasm of differentiated smooth-muscle cells. In overlay assays with portal-vein homogenates, CaM binds predominantly to the MARCKS-containing band. MARCKS is abundant in portal-vein smooth muscle ( approximately 16 microM) in comparison to total CaM ( approximately 40 microM). Confocal images indicate that calmodulin and MARCKS co-distribute in unstimulated freshly dissociated smooth-muscle cells and are co-targeted simultaneously to the cell interior upon depolarization. Protein-kinase-C (PKC) activation triggers a translocation of CaM that precedes that of MARCKS and causes multisite, sequential MARCKS phosphorylation. MARCKS immunoprecipitates with CaM in a stimulus-dependent manner. A synthetic MARCKS effector domain (ED) peptide labelled with a photoaffinity probe cross-links CaM in smooth-muscle tissue in a stimulus-dependent manner. Both cross-linking and immunoprecipitation increase with increased Ca(2+) concentration, but decrease with PKC activation. Introduction of a nonphosphorylatable MARCKS decoy peptide blocks the PKC-mediated targeting of CaM. These results indicate that MARCKS is a significant, PKC-releasable reservoir of CaM in differentiated smooth muscle and that it contributes to CaM signalling by modulating the intracellular distribution of CaM.

Inhibition by Rho-kinase and protein kinase C of myosin phosphatase is involved in thrombin-induced shape change of megakaryocytic leukemia cell line UT-7/TPO.

Thrombin induced a shape change of UT-7/TPO, a thrombopoietin-dependent human megakaryocytic cell line. Expression of myosin light chain (MLC) kinase was negligible in UT-7/TPO cells, while Rho-kinase and protein kinase C (PKC) were detected. Thrombin stimulated both monophosphorylation at Ser19 and diphosphorylation at Thr18 and Ser19 of 20 kDa MLC, as well as phosphorylation of myosin-binding subunit (MBS) and PKC-potentiated inhibitory phosphoprotein of myosin phosphatase (CPI). The Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-(1-aminoethyl)-N-(4-phynidyl) cyclohexane-carboxamide dihydrochloride, monohydrade] strongly inhibited thrombin-induced shape change, MBS phosphorylation, and mono- and diphosphorylation of MLC. The PKC inhibitor GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) partially inhibited thrombin-induced shape change and MLC diphosphorylation even at the concentration that completely inhibited thrombin-induced CPI phosphorylation. In shape-changed UT-7/TPO cells induced by thrombin, phosphorylated MBS and CPI were colocalized with diphosphorylated MLC at pseudopods, whereas monophosphorylated MLC was mainly located in the cortical region. The accumulation of diphosphorylated MLC was blocked by preincubation with either Y-27632 or GF109203X. These results suggest that Rho-kinase is responsible for the induction of MLC phosphorylation in thrombin-induced shape change of UT-7/TPO cells and that myosin phosphatase inactivation through Rho-kinase-MBS and PKC-CPI pathways could be necessary for enhancement of MLC diphosphorylation which promote the pseudopod formation.

Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts.

We examined the role of regulatory myosin light chain (MLC) phosphorylation of myosin II in cell migration of fibroblasts. Myosin light chain kinase (MLCK) inhibition blocked MLC phosphorylation at the cell periphery, but not in the center. MLCK-inhibited cells did not assemble zyxin-containing adhesions at the periphery, but maintained focal adhesions in the center. They generated membrane protrusions all around the cell, turned more frequently, and migrated less effectively. In contrast, Rho-associated kinase (ROCK) inhibition blocked MLC phosphorylation in the center, but not at the periphery. ROCK-inhibited cells assembled zyxin-containing adhesions at the periphery, but not focal adhesions in the center. They moved faster and more straight. On the other hand, inhibition of myosin phosphatase increased MLC phosphorylation and blocked peripheral membrane ruffling, as well as turnover of focal adhesions and cell migration. Our results suggest that myosin II activated by MLCK at the cell periphery controls membrane ruffling, and that the spatial regulation of MLC phosphorylation plays critical roles in controlling cell migration of fibroblasts.

Prostaglandin E2 is an enhancer of interleukin-1beta-induced expression of membrane-associated prostaglandin E synthase in rheumatoid synovial fibroblasts.

OBJECTIVE: Membrane-associated prostaglandin E synthase (mPGES) is a recently identified terminal enzyme of the arachidonic acid cascade, which converts PGH(2) to PGE(2) in rheumatoid arthritis synovial fibroblasts (RASFs). This study was undertaken to investigate factors regulating the expression of mPGES. METHODS: RASFs were treated with interleukin-1beta (IL-1beta), indomethacin, NS-398, rofecoxib, or meloxicam. The effects of PGE(2) and selective agonists for PGE(2) receptor subtypes (EP1, EP2, EP3, and EP4) were also studied. Expression of mPGES messenger RNA (mRNA) and protein was measured by Northern and Western blot analysis, respectively. EP receptor mRNA expression in RASFs was determined by reverse transcriptase-polymerase chain reaction. Production of PGE(2) and cAMP was measured by enzyme-linked immunosorbent assay. RESULTS: The enhanced expression of mPGES mRNA and protein in IL-1beta-stimulated RASFs was attenuated by the addition of indomethacin, NS-398, rofecoxib, or meloxicam. This reduction of expression was reversed by PGE(2). IL-1beta-induced PGES activity, measured by conversion of PGH(2) to PGE(2), was decreased by rofecoxib. EP2 and EP4 receptor mRNA was detected in RASFs. EP2 and EP4 agonists, as well as PGE(2), restored the inhibitory effect of rofecoxib on mPGES expression. The effect of PGE(2) was mimicked by forskolin, a direct activator of adenylate cyclase. Intracellular cAMP was increased by IL-1beta and was inhibited by rofecoxib. CONCLUSION: Enhancement of mPGES expression by PGE(2) via the EP2/EP4 receptors with an increase in cAMP may play an important role in articular inflammation in patients with RA. It also seems that cyclooxygenase 2 (COX-2) inhibitors decrease PGE(2) production not only by direct inhibition of COX-2, but also by reducing mPGES expression in activated RASFs.

Synapsin I is phosphorylated at Ser603 by p21-activated kinases (PAKs) in vitro and in PC12 cells stimulated with bradykinin.

The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser(603) residue (site 3) is considered to be a pivotal site targeted by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser(603) residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKII-involved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser(603) without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser(603)-synapsin I (pS603-Syn I-Ab) and phospho-Thr(286/287)-CaMKII. The bradykinin-evoked phosphorylation of Ser(603) was not suppressed by the CaMKII inhibitor KN62, whereas high KCl-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser(603) kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca(2+)/calmodulin-independent Ser(603) kinase activity on the DEAE column chromatography of bovine brain homogenate and PC12 cell lysate, respectively, two of which were purified and identified by amino acid sequence of proteolytic fragments as p21-activated kinase (PAK) 1 and PAK3. The immunoprecipitants from bovine brain homogenate with anti-PAK1 and PAK3 antibodies incorporated (32)P into synapsin I in a Cdc42/GTPgammaS-dependent manner, and its phosphorylation site was confirmed as Ser(603) using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser(603) residue in the presence of Cdc42/GTPgammaS. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat alphaPAK (PAK1) in PC12 cells evokes the phosphorylation of Ser(603) even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative alphaPAK quenches the phosphorylation. These results raise the possibility that Ser(603) on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.