The present studies sought to determine whether the lethality of the drug combination [sorafenib + sildenafil] could be enhanced by the anti-inflammatory agent celecoxib, using ovarian cancer and other tumor cell lines as models. Also, in a dose dependent fashion celecoxib enhanced [sorafenib + sildenafil] lethality in multiple ovarian cancer cell lines. In a dose dependent fashion celecoxib enhanced the ability of [sorafenib + sildenafil] to reduce expression of multiple chaperone proteins in parallel with lower levels of the drug efflux pumps ABCB1 and ABCG2. Over-expression of GRP78 and HSP27 maintained pump expression in the presence of drugs. Cell killing by the 3 drug combination was mediated by mitochondrial / caspase 9 –dependent apoptotic signaling and by RIP-1 / caspases 2 and 4 / AIF –dependent necroptotic signaling. Pre-treatment of intrinsically resistant primary ovarian cancer cells with [celecoxib + sorafenib + sildenafil] significantly enhanced tumor cell killing by a subsequent cisplatin exposure. Similar data were obtained in some cancer cell lines, but not all, using the related platinum containing drugs, oxaliplatin and carboplatin. As our prior publications have also validated in vivo the combinations of [celecoxib + sildenafil] and [sorafenib + sildenafil] as cytotoxic to multiple tumor cell types, combined with the present findings, we would argue that the combination of celecoxib/sorafenib/sildenafil should be explored in a new phase I trial in ovarian cancer.
We examined the interaction between OSU‐03012 (also called AR‐12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose‐regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU‐03012 to kill stem‐like GBM cells. Treatment of cells with OSU‐03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK‐eIF2α‐ATF4‐CHOP signaling and was blocked by GRP78 over‐expression. In vivo OSU‐03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU‐03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over‐expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU‐03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU‐03012/sildenafil lethality. J. Cell. Physiol. 230: 1982–1998, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi‐kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug‐treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged ‘rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre‐treatment with the ‘Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. ‘Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of ‘Rafenib drugs/pazopanib restored antibiotic sensitivity in pan‐antibiotic resistant bacteria including multiple strains of bla
Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections. J. Cell. Physiol. 230: 2552–2578, 2015. © 2015 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.
We determined whether the multi‐kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as Viagra (sildenafil) to kill tumor cells. PDE5 and PDGFRα/β were over‐expressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death, regardless of whether cells were grown in 10 or 100% human serum. Knock down of PDE5 or of PDGFRα/β recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. Inhibition of CD95/FADD/caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK‐1, Beclin1, or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K, and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas re‐expression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib/regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. From multiplex assays on tumor tissue and plasma, we discovered that increased FGF levels and ERBB1 and AKT phosphorylation were biomarkers that were directly associated with lower levels of cell killing by ‘rafenib + sildenafil. Our data are now being translated into the clinic for further determination as to whether this drug combination is a useful anti‐tumor therapy for solid tumor patients. J. Cell. Physiol. 230: 2281–2298, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.
To determine if combination treatment with pemetrexed and sorafenib is safe and tolerable in patients with advanced solid tumors.
Thirty-seven patients were enrolled and 36 patients were treated (24 in cohort A; 12 in cohort B). The cohort A dose schedule resulted in problematic cumulative toxicity, while the cohort B dose schedule was found to be more tolerable. The maximum tolerated dose (MTD) was pemetrexed 750 mg/m2 every 14 days with oral sorafenib 400 mg given twice daily on days 1–5. Because dosing delays and modifications were associated with the MTD, the recommended phase II dose was declared to be pemetrexed 500 mg/m2 every 14 days with oral sorafenib 400 mg given twice daily on days 1–5. Thirty-three patients were evaluated for antitumor activity. One complete response and 4 partial responses were observed (15% overall response rate). Stable disease was seen in 15 patients (45%). Four patients had a continued response at 6 months, including 2 of 5 patients with triple-negative breast cancer.
A phase I trial employing a standard 3 + 3 design was conducted in patients with advanced solid tumors. Cohort A involved a novel dose escalation schema exploring doses of pemetrexed every 14 days with continuous sorafenib. Cohort B involved a modified schedule of sorafenib dosing on days 1–5 of each 14-day pemetrexed cycle. Radiographic assessments were conducted every 8 weeks.
Pemetrexed and intermittent sorafenib therapy is a safe and tolerable combination for patients, with promising activity seen in patients with breast cancer.
clinical trial; pemetrexed; solid tumors; sorafenib
dimethyl fumarate (DMF); monomethyl fumarate (MMF); Tecfidera; ruxolitinib; Jakafi; glioblastoma multiforme; oncology
We have recently demonstrated that multi-kinase inhibitors such as sorafenib and pazopanib can suppress the detection of chaperones by in situ immuno-fluorescence, which is further enhanced by phosphodiesterase 5 inhibitors. Sorafenib and pazopanib inhibited the HSP90 ATPase activity with IC50 values of ~1.0 μM and ~75 nM, respectively. Pazopanib docked in silico with two possible poses into the HSP90 ATP binding pocket. Pazopanib and sildenafil combined to reduce the total protein levels of HSP1H/p105 and c-MYC and to reduce their co-localization. Sorafenib/pazopanib combined with sildenafil in a [GRP78+HSP27] –dependent fashion to: (i) profoundly activate an eIF2α/Beclin1 pathway; (ii) profoundly inactivate mTOR and increase ATG13 phosphorylation, collectively resulting in the formation of toxic autophagosomes. In a fresh PDX isolate of NSCLC combined knock down of [ERBB1+ERBB3] or use of the ERBB1/2/4 inhibitor afatinib altered cell morphology, enhanced ATG13 phosphorylation, inactivated NFκB, and further enhanced [sorafenib/pazopanib + sildenafil] lethality. Identical data to that with afatinib were obtained knocking down PI3K p110α/β or using buparlisib, copanlisib or the specific p110α inhibitor BYL719. Afatinib adapted NSCLC clones were resistant to buparlisib or copanlisib but were more sensitive than control clones to [sorafenib + sildenafil] or [pazopanib + sildenafil]. Lapatinib significantly enhanced the anti-tumor effect of [regorafenib + sildenafil] in vivo; afatinib and BYL719 enhanced the anti-tumor effects of [sorafenib + sildenafil] and [pazopanib] in vivo, respectively.
sorafenib; pazopanib; chaperones; ERBB; PI3K
There is an urgent need to understand the molecular mechanisms by which sustained exposure to low-dose environmental chemical mixtures promotes carcinogenesis. This Halifax Project review specifically examines the effects of environmental chemicals on the cancer hallmark of evading growth suppression.
As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.
In the completed phase I trial NCT01450384 combining the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib it was observed that 20 of 33 patients had prolonged stable disease or tumor regression, with one complete response and multiple partial responses. The pre-clinical studies in this manuscript were designed to determine whether [pemetrexed + sorafenib] –induced cell killing could be rationally enhanced by additional signaling modulators. Multiplex assays performed on tumor material that survived and re-grew after [pemetrexed + sorafenib] exposure showed increased phosphorylation of ERBB1 and of NFκB and IκB; with reduced IκB and elevated G-CSF and KC protein levels. Inhibition of JAK1/2 downstream of the G-CSF/KC receptors did not enhance [pemetrexed + sorafenib] lethality whereas inhibition of ERBB1/2/4 using kinase inhibitory agents or siRNA knock down of ERBB1/2/3 strongly promoted killing. Inhibition of ERBB1/2/4 blocked [pemetrexed + sorafenib] stimulated NFκB activation and SOD2 expression; and expression of IκB S32A S36A significantly enhanced [pemetrexed + sorafenib] lethality. Sorafenib inhibited HSP90 and HSP70 chaperone ATPase activities and reduced the interactions of chaperones with clients including c-MYC, CDC37 and MCL-1. In vivo, a 5 day transient exposure of established mammary tumors to lapatinib or vandetanib significantly enhanced the anti-tumor effect of [pemetrexed + sorafenib], without any apparent normal tissue toxicities. Identical data to that in breast cancer were obtained in NSCLC tumors using the ERBB1/2/4 inhibitor afatinib. Our data argue that the combination of pemetrexed, sorafenib and an ERBB1/2/4 inhibitor should be explored in a new phase I trial in solid tumor patients.
pemetrexed; sorafenib; ERBB1; PTEN
Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged ‘rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the ‘Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. ‘Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of ‘Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc
Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections.
We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality.
We determined whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as Viagra (sildenafil) to kill tumor cells. PDE5 and PDGFRα/β were over-expressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death, regardless of whether cells were grown in 10 or 100% human serum. Knock down of PDE5 or of PDGFRα/β recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. Inhibition of CD95/FADD/caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK-1, Beclin1, or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K, and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas re-expression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib/regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. From multiplex assays on tumor tissue and plasma, we discovered that increased FGF levels and ERBB1 and AKT phosphorylation were biomarkers that were directly associated with lower levels of cell killing by ‘rafenib + sildenafil. Our data are now being translated into the clinic for further determination as to whether this drug combination is a useful anti-tumor therapy for solid tumor patients.
We generated afatinib resistant clones of H1975 lung cancer cells by transient exposure of established tumors to the drug and collected the re-grown tumors. Afatinib resistant H1975 clones did not exhibit any additional mutations in proto-oncogenes when compared to control clones. Afatinib resistant H1975 tumor clones expressed less PTEN than control clones and in afatinib resistant clones this correlated with increased basal SRC Y416, ERBB3 Y1289, AKT T308 and mTOR S2448 phosphorylation, decreased expression of ERBB1, ERBB2 and ERBB3 and increased total expression of c-MET, c-KIT and PDGFRβ. Afatinib resistant clones were selectively killed by knock down of [ERBB3 + c-MET + c-KIT] but not by the individual or doublet knock down combinations. The combination of the ERBB1/2/4 inhibitor afatinib with the SRC family inhibitor dasatinib killed afatinib resistant H1975 cells in a greater than additive fashion; other drugs used in combination with dasatinib such as sunitinib, crizotinib and amufatinib were less effective. [Afatinib + dasatinib] treatment profoundly inactivated ERBB3, AKT and mTOR in the H1975 afatinib resistant clones and increased ATG13 S318 phosphorylation. Knock down of ATG13, Beclin1 or eIF2α strong suppressed killing by [ERBB3 + c-MET + c-KIT] knock down, but were only modestly protective against [afatinib + dasatinib] lethality. Thus afatinib resistant H1975 NSCLC cells rely on ERBB1- and SRC-dependent hyper-activation of residual ERBB3 and elevated signaling, due to elevated protein expression, from wild type c-MET and c-KIT to remain alive. Inhibition of ERBB3 signaling via both blockade of SRC and ERBB1 results in tumor cell death.
H1975; ERBB1 T790M L858R; afatinib resistance; dasatinib; ERBB3+c-MET+c-KIT
We determined whether the myelofibrosis drug ruxolitinib, an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could interact with the multiple sclerosis drug dimethyl-fumarate (DMF) to kill tumor cells; studies used the in vivo active form of the drug, mono-methyl fumarate (MMF). Ruxolitinib interacted with MMF to kill brain, breast, lung and ovarian cancer cells, and enhanced the lethality of standard of care therapies such as paclitaxel and temozolomide. MMF also interacted with other FDA approved drugs to kill tumor cells including Celebrex® and Gilenya®. The combination of [ruxolitinib + MMF] inactivated ERK1/2, AKT, STAT3 and STAT5; reduced expression of MCL-1, BCL-XL, SOD2 and TRX; increased BIM expression; decreased BAD S112 S136 phosphorylation; and enhanced pro-caspase 3 cleavage. Expression of activated forms of STAT3, MEK1 or AKT each significantly reduced drug combination lethality; prevented BAD S112 S136 dephosphorylation and decreased BIM expression; and preserved TRX, SOD2, MCL-1 and BCL-XL expression. The drug combination increased the levels of reactive oxygen species in cells, and over-expression of TRX or SOD2 prevented drug combination tumor cell killing. Over-expression of BCL-XL or knock down of BAX, BIM, BAD or apoptosis inducing factor (AIF) protected tumor cells. The drug combination increased AIF : HSP70 co-localization in the cytosol but this event did not prevent AIF : eIF3A association in the nucleus.
ruxolitinib; JAK-STAT; DMF; tecfidera; ROS
The present studies sought to determine whether the anti-folate pemetrexed (Alimta) and the sphingosine-1-phosphate receptor modulator FTY720 (Fingolimod, Gilenya) interacted to kill tumor cells. FTY720 and pemetrexed interacted in a greater than additive fashion to kill breast, brain and colorectal cancer cells. Loss of p53 function weakly enhanced the toxicity of FTY720 whereas deletion of activated RAS strongly or expression of catalytically inactive AKT facilitated killing. Combined drug exposure reduced the activity of AKT, p70 S6K and mTOR and activated JNK and p38 MAPK. Expression of activated forms of AKT, p70 S6K and mTOR or inhibition of JNK and p38 MAPK suppressed the interaction between FTY720 and pemetrexed. Treatment of cells with FTY720 and pemetrexed increased the numbers of early autophagosomes but not autolysosomes, which correlated with increased LC3II processing and increased p62 levels, suggestive of stalled autophagic flux. Knock down of ATG5 or Beclin1 suppressed autophagosome formation and cell killing. Knock down of ceramide synthase 6 suppressed autophagosome production and cell killing whereas knock down of ceramide synthase 2 enhanced vesicle formation and facilitated death. Collectively our findings argue that pemetrexed and FTY720 could be a novel adjunct modality for breast cancer treatment.
FTY720; Gilenya; Pemetrexed; Alimta; ceramide; S1P; autophagy; CerS2; CerS6
We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone – chaperone and chaperone – client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 –dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 –induced activation of ER stress signaling and maintained mTOR activity; AR-12 –mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.
OSU-03012; sorafenib; pazopanib; chaperones; ATPase
The chaperone protein HSPA5/Dna K is conserved throughout evolution from higher eukaryotes down to prokaryotes. The celecoxib derivative OSU-03012 (also called AR-12) interacts with Viagra or Cialis in eukaryotic cells to rapidly reduce HSPA5 levels as well as blunt the functions of many other chaperone proteins. Because multiple chaperones are modulated in eukaryotes, the expression of cell surface virus receptors is reduced and because HSPA5 in blocked viruses cannot efficiently replicate. Because DnaK levels are reduced in prokaryotes by OSU-03012, the levels of DnaK chaperone proteins such as Rec A decline, which is associated with bacterial cell death and a resensitization of so-called drug-resistant superbugs to standard of care antibiotics. In Alzheimer's disease, HSPA5 has been shown to play a supportive role for the progression of tau phosphorylation and neurodegeneration. Thus, in eukaryotes, HSPA5 represents a target for anticancer, antiviral, and anti-Alzheimer's therapeutics and in prokaryotes, DnaK and bacterial phosphodiesterases represent novel antibiotic targets that should be exploited in the future by pharmaceutical companies.
We previously reported that Sildenafil enhances apoptosis and antitumor efficacy of doxorubicin (DOX) while attenuating its cardiotoxic effect in prostate cancer. In the present study, we investigated the mechanism by which sildenafil sensitizes DOX in killing of prostate cancer (PCa) cells, DU145. The death receptor Fas (APO-1 or CD95) induces apoptosis in many carcinoma cells, which is negatively regulated by anti-apoptotic molecules such as FLIP (Fas-associated death domain (FADD) interleukin-1-converting enzyme (FLICE)-like inhibitory protein). Co-treatment of PCa cells with sildenafil and DOX for 48 hours showed reduced expression of both long and short forms of FLIP (FLIP-L and -S) as compared to individual drug treatment. Over-expression of FLIP-s with an adenoviral vector attentuated the enhanced cell-killing effect of DOX and sildenafil. Colony formation assays also confirmed that FLIP-S over-expression inhibited the DOX and sildenafil-induced synergistic killing effect as compared to the cells infected with an empty vector. Moreover, siRNA knock-down of CD95 abolished the effect of sildenafil in enhancing DOX lethality in cells, but had no effect on cell killing after treatment with a single agent. Sildenafil co-treatment with DOX inhibited DOX-induced NF-κB activity by reducing phosphorylation of IκB and nuclear translocation of the p65 subunit, in addition to down regulation of FAP-1 (Fas associated phosphatase-1, a known inhibitor of CD95-mediated apoptosis) expression. This data provides evidence that the CD95 is a key regulator of sildenafil and DOX mediated enhanced cell death in prostate cancer.
PDE5; doxorubicin; CD95; FLIP; prostate cancer
We determined whether the approved myelofibrosis drug ruxolitinib (Jakafi®), an inhibitor of Janus kinases 1/2 (JAK1 and JAK2), could be repurposed as an anti-cancer agent for solid tumors. Ruxolitinib synergistically interacted with dual ERBB1/2/4 inhibitors to kill breast as well as lung, ovarian and brain cancer cells. Knock down of JAK1/2 or of ERBB1/2/3/4 recapitulated on-target drug effects. The combination of (ruxolitinib + ERBB1/2/4 inhibitor) rapidly inactivated AKT, mTORC1, mTORC2, STAT3, and STAT5, and activated eIF2α. In parallel, the drug combination reduced expression of MCL-1, BCL-XL, HSP90, HSP70, and GRP78, and increased expression of Beclin1. Activated forms of STAT3, AKT, or mTOR prevented the drug-induced decline in BCL-XL, MCL-1, HSP90, and HSP70 levels. Over-expression of chaperones maintained AKT/mTOR activity in the presence of drugs and protected tumor cells from the drug combination. Expression of dominant negative eIF2α S51A prevented the increase in Beclin1 expression and protected tumor cells from the drug combination. Loss of mTOR activity was associated with increased ATG13 S318 phosphorylation and with autophagosome formation. Autophagosomes initially co-localized with mitochondria and subsequently with lysosomes. Knock down of Beclin1 suppressed: drug-induced mitophagy; the activation of the toxic BH3 domain proteins BAX and BAK; and tumor cell killing. Knock down of apoptosis-inducing factor (AIF) protected tumor cells from the drug combination, whereas blockade of caspase 9 signaling did not. The drug combination released AIF into the cytosol and increased nuclear AIF: eIF3A co-localization. A 4-day transient exposure of orthotopic tumors to (ruxolitinib + afatinib) profoundly reduced mammary tumor growth over the following 35 days. Re-grown tumors exhibited high levels of BAD S112 phosphorylation and activation of ERK1/2 and NFκB. Our data demonstrate that mitophagy is an essential component of (ruxolitinib + ERBB inhibitor) lethality and that this drug combination should be explored in a phase I trial in solid tumor patients.
ruxolitinib; JAK1/2; afatinib; ERBB1; mitophagy; chaperone; AIF
The present studies were to determine whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with the ERBB1/ERBB2 inhibitor lapatinib to kill CNS tumor cells. In multiple CNS tumor cell types sorafenib and lapatinib interacted in a greater than additive fashion to cause tumor cell death. Tumor cells lacking PTEN, and anoikis or lapatinib resistant cells were as sensitive to the drug combination as cells expressing PTEN or parental cells, respectively. Similar data were obtained using regorafenib. Treatment of brain cancer cells with [sorafenib + lapatinib] enhanced radiation toxicity. The drug combination increased the numbers of LC3-GFP vesicles; this correlated with a reduction in endogenous LC3II, and p62 and LAMP2 degradation. Knock down of Beclin1 or ATG5 significantly suppressed drug combination lethality. Expression of c-FLIP-s, BCL-XL or dominant negative caspase 9 reduced drug combination toxicity; knock down of FADD or CD95 was protective. Expression of both activated AKT and activated MEK1 or activated mTOR was required to strongly suppress drug combination lethality. As both lapatinib and sorafenib are FDA approved agents, our data argue for further determination as to whether lapatinib and sorafenib is a useful glioblastoma therapy.
Sorafenib; Lapatinib; Autophagy; Glioma; AKT; ERK1/2; mTOR; PTEN; p70 S6K; Necrosis
First identified almost two decades ago as a novel gene differentially expressed in human melanoma cells induced to terminally differentiate, MDA-7/IL-24 has since shown great potential as an anti-cancer gene. MDA-7/IL24, a secreted protein of the IL-10 family, functions as a cytokine at normal physiological levels and is expressed in tissues of the immune system. At supra-physiological levels, MDA-7/IL-24 plays a prominent role in inhibiting tumor growth, invasion, metastasis and angiogenesis and was recently shown to target tumor stem/initiating cells for death. Much of the attention focused on MDA-7/IL-24 originated from the fact that it can selectively induce cell death in cancer cells without affecting normal cells. Thus, this gene originally shown to be associated with melanoma cell differentiation has now proven to be a multi-functional protein affecting a broad array of cancers. Moreover, MDA-7/IL-24 has proven efficacious in a Phase I/II clinical trial in humans with multiple advanced cancers. As research in the field progresses, we will unravel more of the functions of MDA-7/IL-24 and define novel ways to utilize MDA-7/IL-24 in the treatment of cancer.
MDA-7; IL-24; Cytokine; Cancer; Apoptosis; Autophagy; Bystander antitumor activity; Cancer terminator virus
The present studies examined the biology of the multiple sclerosis drug dimethyl-fumarate (DMF) or its in vivo breakdown product and active metabolite mono-methyl-fumarate (MMF), alone or in combination with proteasome inhibitors, in primary human glioblastoma (GBM) cells. MMF enhanced velcade and carfilzomib toxicity in multiple primary GBM isolates. Similar data were obtained in breast and colon cancer cells. MMF reduced the invasiveness of GBM cells, and enhanced the toxicity of ionizing radiation and temozolomide. MMF killed freshly isolated activated microglia which was associated with reduced IL-6, TGFβ and TNFα production. The combination of MMF and the multiple sclerosis drug Gilenya further reduced both GBM and activated microglia viability and cytokine production. Over-expression of c-FLIP-s or BCL-XL protected GBM cells from MMF and velcade toxicity. MMF and velcade increased plasma membrane localization of CD95, and knock down of CD95 or FADD blocked the drug interaction. The drug combination inactivated AKT, ERK1/2 and mTOR. Molecular inhibition of AKT/ERK/mTOR signaling enhanced drug combination toxicity whereas molecular activation of these pathways suppressed killing. MMF and velcade increased the levels of autophagosomes and autolysosomes and knock down of ATG5 or Beclin1 protected cells. Inhibition of the eIF2α/ATF4 arm or the IRE1α/XBP1 arm of the ER stress response enhanced drug combination lethality. This was associated with greater production of reactive oxygen species and quenching of ROS suppressed cell killing.
The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors, and to determine the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knock down of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012, and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase 9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase 8 inhibitor c-FLIP-s, or knock down of death receptor CD95 or the death receptor – caspase 8 linker protein FADD, suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knock down of the autophagy regulatory proteins Beclin1 or ATG5 protected cells from OSU-03012 and of [OSU-03012 + PDE5 inhibitor] toxicity. Knock down of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor] –induced JNK activation and inhibition of JNK suppressed the elevated killing caused by IRE1 knock down. Knock down of CD95 blunted JNK activation. Collectively our data demonstrates that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in GBM cells.
Low-dose exposures to common environmental chemicals that are deemed safe individually may be combining to instigate carcinogenesis, thereby contributing to the incidence of cancer. This risk may be overlooked by current regulatory practices and needs to be vigorously investigated.
Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety ‘Mode of Action’ framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology.
Receptor tyrosine kinases play important roles in the biology of many tumor cell types. In approximately 10% of non-small cell lung cancer (NSCLC) patients mutational activation of the epidermal growth factor receptor (EGFR) results in tumor cells that are exquisitely addicted to signaling by this receptor.1 Thus expression of mutant active EGFR but in general not wild-type EGFR predisposes NSCLC cells to inhibitors of EGFR/ErbB2. Use of EGFR inhibitory agents such as gefitinib for this subset of NSCLC patients causes tumor regression and disease stabilization for 12–18 mo, after which tumor cells become resistant to the drug.2 Initial studies identified a second mutation within the EGFR, which results in the resistance of the tyrosine kinase to gefitinib, as a major cause of reduced tumor control.3 This has resulted in the development of newer EGFR inhibitors, e.g., afatinib, which inhibited double mutant EGFR.4 In a subset of these patients, however, resistance to gefitinib was not associated with EGFR mutations.5 Clearly, other mechanisms of gefitinib resistance must be at play.
c-Met; ALK; EGFR; mTOR; targeted cancer therapy; combination therapy