Sorafenib combined vitamin K induces apoptosis in human pancreatic cancer cell lines through RAF/MEK/ERK and c‐Jun NH2‐terminal kinase pathways

Apoptosis has been shown to be induced by many agents, including the clinically useful Sorafenib and K vitamins (VKs). Since few agents have activity against pancreas cancer cell growth, we evaluated the role of naturally occurring K vitamins and Sorafenib both independently and together on the growth in culture of pancreas adenocarcinoma cell lines, including PL‐5, PANC‐1, and MIA PaCa‐2. We found that when a K vitamin was combined with Sorafenib, the dose of Sorafenib required for growth inhibition was substantially reduced. Furthermore, growth could be inhibited at doses of each VK plus Sorafenib in combination that were ineffective when used alone. This effect was seen using vitamins K1, K2, and K5. The combination of VK1 plus Sorafenib‐induced apoptosis, as determined by both FACS and TUNEL staining. Phospho‐ERK and Bcl‐2 levels were decreased, but not levels of other bcl‐2 family members. Cleavage of caspases 3 and 8, PARP and Bid were all induced by this combination. Vitamin K1 plus Sorafenib combination also resulted in elevated levels of activated c‐Jun N‐terminal kinase (JNK) and its substrates c‐Jun and FasL. JNK inhibition partly antagonized the induction of apoptosis. Thus, combination VK1 plus Sorafenib strongly induced growth inhibition and apoptosis in pancreas cancer cells, involving both inhibition of the RAF/MEK/ERK pathway as well as activation of the JNK, c‐Jun and FasL apoptotic pathway. Since both agents are available for human use, the combination is attractive for evaluation against pancreas cancer growth in vivo. J. Cell. Physiol. 224:112–119, 2010 © 2010 Wiley‐Liss, Inc.     

Role of RAF/MEK/ERK pathway,p‐STAT‐3 and Mcl‐1 in sorafenib activity in human pancreatic cancer cell lines

Sorafenib is a multikinase inhibitor that has shown promising therapeutic results in different tumor histotypes, both as a single agent or in combination with other treatments. We analyzed the in vitro activity of sorafenib in pancreatic cancer, one of the most lethal and chemo‐radio‐resistant tumors, using four human pancreatic cancer cell lines (t3m4, Capan 1, Capan 2, and MiaPaca 2), characterized by different K‐ras gene status and RAF/MEK/ERK profile. Sorafenib exerted a strong anti‐proliferative effect independently of RAS/RAF/MEK/ERK and induced various degrees of apoptosis in the cell lines. The mechanisms involved were explored in detail in t3m4 and Capan 1, in which sorafenib induced the highest and lowest levels of apoptosis, respectively. In t3m4, the RAF/AKT/STAT‐3 rather than the RAF/MEK/ERK pathway was involved, whereas in Capan 1 cells there was a strong decrease in pMEK and pERK which was not accompanied by an important reduction in RAF, AKT, and STAT‐3 proteins or in their phosphorylation. Moreover, U0126‐induced MEK inhibition did not induce apoptosis in any cell line, reinforcing the hypothesis of a MEK/ERK‐independent mechanism of sorafenib activity. Mcl‐1 appears to play a crucial role in sorafenib‐induced apoptosis. In fact, both protein and mRNA were downregulated in t3m4 and upregulated in Capan 1, in which siRNA‐induced silencing resulted in the same level of apoptosis as observed in t3m4. Our results show that sorafenib exerts anti‐proliferative and pro‐apoptotic activity in pancreatic cancer cells. Used singly or in combination with other drugs, it could therefore represent valid treatment for pancreatic cancer. J. Cell. Physiol. 220: 214–221, 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of doxorubicin-induced autophagy in hepatocellular carcinoma Hep3B cells by sorafenib--the role of extracellular signal-regulated kinase counteraction

A multikinase inhibitor of the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, sorafenib, is increasingly being used in the management of hepatocellular carcinoma, and its combination with conventional chemotherapeutics has stimulated particular interest. Although the combination of sorafenib with doxorubicin (DOX) is presently being investigated in a phase III randomized trial, little is known about the molecular mechanisms of their interaction. Because DOX causes cell death through upregulation of the MEK/ERK pathway, and sorafenib has an opposite influence on the same cascade, we hypothesized that co-treatment with these drugs may lead to an antagonistic effect. DOX treatment arrested proliferation and induced autophagic cell death in Hep3B cells, whereas apoptotic changes were not conspicuous. Sorafenib alone affected viability and caused massive mitochondrial degradation. However, when added together with DOX, sorafenib facilitated cell cycle progression, increased survival, and reduced autophagy. To evaluate the molecular mechanisms of this phenomenon, we examined the expression of ERK1/2, protein kinase B (Akt), and cyclin D1, as well as the members of Bcl-2 family. ERK1/2 activation induced by DOX was suppressed by sorafenib. Similarly, ERK targeting with the selective inhibitor U0126 impaired DOX-induced toxicity. Treatment with sorafenib, either alone or in combination with DOX, resulted in Akt activation. The role of sorafenib-induced degradation of cyclin D1 in the suppression of DOX efficiency is discussed. In conclusion, MEK/ERK counteraction, stimulation of survival via Akt and dysregulation of cyclin D1 could contribute to the escape from DOX-induced autophagy and thus promote cancer cell survival. The use of MEK/ERK inhibitors in combination with chemotherapeutics, intended to enhance anticancer efficacy, requires the consideration of possible antagonistic effects.     

VEGFR,RET, and RAF/MEK/ERK Pathway Take Part in the Inhibition of Osteosarcoma MG63 Cells with Sorafenib Treatment

Osteosarcoma (OS) is the leading primary malignant bone tumor in children and young adults. It is response for a high mortality rate. Nowadays, few researches have been performed on sorafenib against OS and no tools are available to guide the use of sorafenib in the OS treatment. In this study, we aim to investigate the effect of sorafenib on OS cell MG63 and figure the potential effective molecular pathway of its function. In the present study, we performed assays of cell proliferation, RT-PCR, and western blot to investigate the effect of sorafenib on OS MG63 cells and to elucidate the molecular actions of sorafenib against RTKs VEGFR2 and RET, as well as MEK/ERK signaling pathway. The present study confirmed that sorafenib could inhibit the proliferation of OS MG63 cells and caused a series of biomolecule effects, including the change of VEGFR2 and ERK gene expression, and the phosphorylation alteration of VEGFR2, RET, and MEK1 proteins. VEGFR2, RET, and MEK/ERK signaling pathway are involved in the pharmacological mechanism of sorafenib. They are potential candidate targets for OS treatment.     

Differential inhibitory effects of two Raf-targeting drugs, sorafenib and PLX4720, on the growth of multidrug-resistant cells

B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade in human cancers, making it an important therapeutic target. Here, we describe the differential effects of two Raf-targeting drugs, sorafenib and PLX4720, on multidrug-resistant v-Ha-ras-transformed cells (Ras-NIH 3T3/Mdr). We demonstrate that the growth of the NIH 3T3/Mdr cell line was affected in a dose-dependent manner more significantly by the pan-Raf inhibitor sorafenib than by the selective mutant B-Raf inhibitor PLX4720. Despite their differential effects on LKB1/AMPK phosphorylation, both sorafenib and PLX4720 inhibited downstream mTOR signaling with concomitant induction of autophagy, implying that the differential effects of sorafenib and PLX4720 on multidrug-resistant cells might not be due to different levels of autophagy and apoptosis. Interestingly, sorafenib caused a dose-dependent increase in rhodamine 123 uptake and retention. More importantly, sorafenib reversed the resistance to paclitaxel in Ras-NIH 3T3/Mdr cells. Moreover, MEK/ERK signaling was hyperactivated by the selective mutant B-Raf inhibitor PLX4720 and inhibited by the pan-Raf inhibitor sorafenib. Our data suggest that sorafenib sensitivity in MDR cells is mediated through the inhibition of P-glycoprotein activity following strong inhibition of Raf/MEK/ERK signaling. Thus, Raf inhibition with sorafenib might be a promising approach to abrogate the multidrug resistance of cancer cells.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

Vitamin D(3)-induced apoptosis of murine squamous cell carcinoma cells. Selective induction of caspase-dependent MEK cleavage and up-regulation of MEKK-1

Vitamin D(3) inhibits cell growth and induces apoptosis in several human cancer lines in vitro and in vivo. However, little is known about the molecular events involved in vitamin D(3)-induced apoptosis. Here, we demonstrate that the growth-promoting/pro-survival signaling molecule mitogen-activated protein kinase kinase (MEK) is cleaved in a caspase-dependent manner in murine squamous cell carcinoma (SCC) cells induced to undergo apoptosis by treatment with vitamin D(3). Cleavage resulted in nearly complete loss of full-length MEK and ERK1/2 phosphorylation. ERK1/2 expression was affected only slightly. The phosphorylation and expression of Akt, a kinase regulating a second cell survival pathway, was also inhibited after treatment with vitamin D(3). However, the pro-apoptotic signaling molecule MEKK-1 was up-regulated in both apoptotic and non-apoptotic cells with greater induction and partial N-terminal proteolysis of MEKK-1 observed in apoptotic cells. In contrast to vitamin D(3), cisplatin and etoposide down-regulated Akt levels only modestly, did not promote significant loss of MEK expression, and did not up-regulate MEKK-1. We propose that vitamin D(3) induces apoptosis in SCC cells by a unique mechanism involving selective caspase-dependent MEK cleavage and up-regulation of MEKK-1. Additional evidence is provided that vitamin D(3)-induced apoptosis may be mediated via p38 MAPK.     

Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis

Sorafenib, a potent multikinase inhibitor, has been recognized as the standard systemic treatment for patients with advanced hepatocellular carcinoma (HCC). However, the direct functional mechanism of tumor lethality mediated by sorafenib remains to be fully characterized, and the precise mechanisms of drug resistance are largely unknown. Here, we showed sorafenib induced both apoptosis and autophagy in human HCC cells through a mechanism that involved endoplasmic reticulum (ER) stress and was independent of the MEK1/2-ERK1/2 pathway. Upregulation of IRE1 signals from sorafenib-induced ER stress was critical for the induction of autophagy. Moreover, autophagy activation alleviated the ER stress-induced cell death. Inhibition of autophagy using either pharmacological inhibitors or essential autophagy gene knockdown enhanced cell death in sorafenib treated HCC cell lines. Critically, the combination of sorafenib with the autophagy inhibitor chloroquine produced more pronounced tumor suppression in HCC both in vivo and in vitro. These findings indicated that both ER stress and autophagy were involved in the cell death evoked by sorafenib in HCC cells. The combination of autophagy modulation and molecular targeted therapy is a promising therapeutic strategy in treatment of HCC.     

MEK blockade overcomes the limited activity of palbociclib in head and neck cancer

Head and neck cancer (HNC) is characterized with multiple aberrations in cell cycle pathways, including amplification of cyclin D1. Palbociclib (PAL), a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has been reported to regulate cell cycle progression in HNC. However, recent studies have revealed the acquired resistance of certain cells to PAL through activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Therefore, we investigated whether the inhibition of MEK/ERK pathway by trametinib (TRA) may overcome the limited efficacy of PAL in HNC. We evaluated the effect of PAL alone and in combination with TRA on the viability of HNC cells, and found that the combination treatment synergistically inhibited the proliferation of HNC cells. The combination treatment induced G0/G1 cell cycle arrest and apoptotic cell death. In particular, apoptosis mediated by the combination treatment was accompanied with an increase in caspase-3 activity and the number of TUNEL-positive apoptotic cells. These results were consistent with the decrease in cell cycle progression and mitogen-activated protein kinase (MAPK) pathway activation. In a xenograft mouse model of HNC, PAL and TRA synergistically inhibited tumor growth and enhanced tumor cell apoptosis, consistent with the increase in the number of TUNEL-positive cells. The anti-proliferative effects were evident in tumor tissues subjected to the combination treatment as compared with those treated with single drug. Taken together, our study demonstrates that the combination of PAL and TRA exerts synergistic anticancer effects and inhibits cell cycle check points and MEK/ERK pathway in HNC, suggestive of their potential application for HNC treatment.     

Inhibition of ligand-independent ERK1/2 activity in kidney proximal tubular cells deprived of soluble survival factors up-regulates Akt and prevents apoptosis

Mouse kidney proximal tubular epithelial (MK-PT) cells die by apoptosis over 7-10 days when deprived of all survival factors. We show here that withdrawal of all survival factors from MK-PT cells is associated with a progressive increase in the activity of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and a progressive decrease in phosphorylated Akt, a kinase critical to cell survival. Pharmacological inhibition of MEK1/2, the immediate upstream kinase for ERK1/2, not only prevented the decrease in phosphorylated Akt, but also prolonged MK-PT cell survival. Inhibition of ERK1/2, by itself, in the absence of any other known survival factors, was as potent as epidermal growth factor in maintaining MK-PT cell viability. ERK1/2 co-immunoprecipitated with Akt in a multimolecular assembly of signaling molecules, containing at a minimum ERK1/2, Akt, Rsk, and 3-phosphoinositide dependent kinase 1 (PDK1). We hypothesize that the kinase Rsk, whose activation requires phosphorylation by both ERK1/2 and PDK1, acts as a bridge bringing ERK1/2 into proximity with PDK1-associated Akt. Although a number of interactions between the Raf-MEK-ERK and PI3K-Akt signaling pathways have been described, our results are the first to show modulation of Akt activity by signaling events originating with ERK1/2. Spontaneous activation of ERK1/2 occurs via MEK1/2 and appears to depend on oxidant stress, accompanying induction of the default pathway of apoptosis. Together, these data suggest that the spontaneous activation of ERK1/2, in the absence of known extracellular stimuli, represents a previously unrecognized major regulatory pathway determining the fate of cells destined to die by the default pathway of apoptosis.     

Epigallocatechin-3-gallate (EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation

Epigallocatechin-3-gallate (EGCG), a tea polyphenol, inhibits the proliferation of many cancer cell lines; however, the antiproliferative mechanism(s) are not well-characterized. The objective of this study is to identify the cellular signaling mechanism(s) responsible for the antiproliferative effects of EGCG in the PC-3 prostate cancer cell line. EGCG inhibited PC-3 cell proliferation in a concentration-dependent manner with an IC(50) value of 39.0 microM, but had no effect on the proliferation of a nontumorigenic prostate epithelial cell line (RWPE-1). Treatment of PC-3 cells with EGCG (0-50 microM) resulted in time and concentration-dependent activation of the extracellular signal-regulated kinase (ERK1/2) pathway. EGCG treatment did not induce ERK1/2 activity in RWPE-1 cells. The activation of ERK1/2 by EGCG was not inhibited using PD98059, a potent inhibitor of mitogen-activated protein kinase kinase (MEK), the immediate upstream kinase responsible for ERK1/2 activation; suggesting a MEK-independent signaling mechanism. Pretreatment of PC-3 cells with a phosphoinositide-3 kinase (PI3K) inhibitor partially reduced both EGCG-induced ERK1/2 activation and the antiproliferative effects of this polyphenol. These results suggest that ERK1/2 activation via a MEK-independent, PI3-K-dependent signaling pathway is partially responsible for the antiproliferative effects of EGCG in PC-3 cells.     

The regulation of ERK and p-ERK expression by cisplatin and sorafenib in gastric cancer cells

Previous studies have reported strong antitumor effects of cisplatin and sorafenib. Our results indicated that cisplatin and sorafenib exhibited anti-tumor effects on gastric cancer cells. They significantly inhibited gastric cell growth and induced apoptosis. They effectively inhibited gastric cancer cell proliferation and induced G0/G1 phase arrest. Western blotting analysis indicated that it also promoted the phosphorylation extracellular signal regulated kinase (p-ERK). Moreover, cisplatin and sorafenib played a synergistic antitumor effect. These results suggested that the antitumor mechanism of cisplatin and sorafenib involved altering the cell cycle and stimulating ERK phosphorylation in the ERK signaling pathway.     

Calcium-activated RAF/MEK/ERK signaling pathway mediates p53-dependent apoptosis and is abrogated by alpha B-crystallin through inhibition of RAS activation

The ocular lens is the only organ that does not develop spontaneous tumor. The molecular mechanism for this phenomenon remains unknown. Through examination of the signaling pathways mediating stress-induced apoptosis, here we presented evidence to show that different from most other tissues in which the extracellular signal-regulated kinases (ERKs) pathway is generally implicated in mediation of survival signals activated by different factors, the RAF/MEK/ERK signaling pathway alone plays a key role in stress-activated apoptosis of lens epithelial cells. Treatment of N/N1003A cells with calcimycin, a calcium mobilizer, activates the RAF/MEK/ERK pathway through RAS, which is indispensable for the induced apoptosis because inhibition of this pathway by either pharmacological drug or dominant negative mutants greatly attenuates the induced apoptosis. Calcimycin also activates p38 kinase and JNK2, which are not involved in calcium-induced apoptosis. Downstream of ERK activation, p53 is essential. Activation of RAF/MEK/ERK pathway by calcimycin leads to distinct up-regulation of p53. Moreover, overexpression of p53 enhances calcimycin-induced apoptosis, whereas inhibition of p53 expression attenuates calcimycin-induced apoptosis. Up-regulation of p53 directly promotes Bax expression, which changes the integrity of mitochondria, leading to release of cytochrome c, activation of caspase-3 and eventually execution of apoptosis. Overexpression of alphaB-crystallin, a member of the small heat-shock protein family, blocks activation of RAS to inhibit ERK1/2 activation, and greatly attenuates calcimycin-induced apoptosis. Together, our results provide 1) a partial explanation for the lack of spontaneous tumor in the lens, 2) a novel signaling pathway for calcium-induced apoptosis, and 3) a novel antiapoptotic mechanism for alphaB-crystallin.     

Inhibition of MEK signaling enhances the ability of cytarabine to induce growth arrest and apoptosis of acute myelogenous leukemia cells

The mitogen-activated protein kinase/ERK kinase (MEK)/ERK pathway was shown to be constitutively activated in a large number of acute myelogenous leukemia (AML) cells, suggesting the important roles of this pro-survival signaling in leukemogenesis and proliferation of AML cells. This study explored the impact of the MEK inhibitor AZD6244 on the effect of cytarabien (AraC), one of the most commonly used anti-leukemia agents, to induce growth arrest and apoptosis of AML cells. AZD6244 effectively blocked AraC-induced MEK/ERK activation and enhanced its ability to induce growth arrest and apoptosis of NB4 and HL60 cells in parallel with induction of DNA damage as measured by detection of γ-H2AX by Western Blot analysis, resulting in enhanced expression of p21 ^waf1 and downregulation of c-Myc and Bcl-xl in these cells. Enhanced induction of apoptosis mediated by combination of AZD6244 and AraC was also shown in freshly isolated AML cells (n = 3). Taken together, concomitant administration of AraC and the inhibitor of MEK/ERK signaling may be useful for treatment of individuals with AML.