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Cancer vaccines that make use of tumor antigens represent a promising therapeutic strategy by stimulating immune responses against tumors to generate long-term anti-tumor immunity. However, vaccines have shown limited clinical efficacy due to inefficient delivery. In this study, we focus on vaccine delivery assisted by nanocomplexes for cancer immunotherapy. Nanocomplex-mediated vaccination can efficiently deliver nucleic acids encoding neoantigens to lymphoid tissues and antigen-presenting cells. Polyethylenimine (PEI) was conjugated with farnesylthiosalicylic acid (FTS) to form micelles. Subsequent interaction with nucleic acids led to formation of polymer/nucleic acid nanocomplexes of well-controlled structure. Tumor transfection via FTS-PEI was much more effective than that by PEI, other PEI derivatives, or naked DNA. Significant numbers of transfected cells were also observed in draining lymph nodes (LNs). In vivo delivery of ovalbumin (OVA; a model antigen) expression plasmid (pOVA) by FTS-PEI led to a significant growth inhibition of the OVA-expressing B16 tumor through presentation of OVA epitopes as well as other epitopes via epitope spreading. Moreover, in vivo delivery of an endogenous melanoma neoantigen tyrosinase-related protein 2 (Trp2) also led to substantial tumor growth inhibition. FTS-PEI represents a promising transfection agent for effective gene delivery to tumors and LNs to mediate effective neoantigen vaccination.

Hui Huang,1,2 Qinhui Liu,1 Ting Zhang,2 Jinhang Zhang,1,2 Jian Zhou,1,2 Xiandan Jing,2 Qin Tang,1 Cuiyuan Huang,1,2 Zijing Zhang,1 Yingnan Zhao,1,2 Guorong Zhang,1,2 Jiamin Yan,1,2 Yan Xia,1,2 Ying Xu,1,2 Jiahui Li,1,2 Yanping Li,1 Jinhan He1,2 1Laboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, People’s Republic of China; 2Department of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, People’s Republic of ChinaCorrespondence: Yanping LiLaboratory of Clinical Pharmacy and Adverse Drug Reaction, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, Sichuan, People’s Republic of ChinaTel +86-28-85164128Email liyanping_512@163.comJinhan HeDepartment of Pharmacy, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu, Sichuan, People’s Republic of ChinaTel +86-28-85426416Email jinhanhe@scu.edu.cnBackground: Renal fibrosis is the common pathway in chronic kidney diseases progression to end-stage renal disease, but to date, no clinical drug for its treatment is approved. It has been demonstrated that the inhibitor of proto-oncogene Ras, farnesylthiosalicylic acid (FTS), shows therapeutic potential for renal fibrosis, but its application was hindered by the water-insolubility and low bioavailability. Hence, in this study, we improved these properties of FTS by encapsulating it into bovine serum albumin nanoparticles (AN-FTS) and tested its therapeutic effect in renal fibrosis.Methods: AN-FTS was developed using a classic emulsification-solvent ultrasonication. The pharmacokinetics of DiD-loaded albumin nanoparticle were investigated in SD rats. The biodistribution and therapeutic efficacy of AN-FTS was assessed in a mouse model of renal fibrosis induced by unilateral ureteral obstruction (UUO).Results: AN-FTS showed a uniform spherical shape with the size of 100.6 ± 1.12 nm and PDI < 0.25. In vitro, AN-FTS displayed stronger inhibitory effects on the activation of renal fibroblasts cells NRK-49F than free FTS. In vivo, AN-FTS showed significantly higher peak concentration and area under the concentration-time curve. After intravenous administration to UUO-induced renal fibrosis mice, AN-FTS accumulated preferentially in the fibrotic kidney, and alleviated renal fibrosis and inflammation significantly more than the free drug. Mechanistically, the improved anti-fibrosis effect of AN-FTS was associated with greater inhibition in renal epithelial-to-mesenchymal transformation process via Ras/Raf1/p38 signaling pathway.Conclusion: The study reveals that AN-FTS is capable of delivering FTS to fibrotic kidney and showed superior therapeutic efficacy for renal fibrosis.Keywords: albumin nanoparticle, epithelial–mesenchymal transition, farnesylthiosalicylic acid, Ras, renal fibrosis

The activity of Ras, a small GTPase protein, is increased in brains with Alzheimer’s disease. The objective of this study was to determine the influence of oligomeric Aβ1-42 on the activation of Ras, and the involvement of the Ras hyperactivity in Aβ1-42-induced deficits in spatial cognition and hippocampal synaptic plasticity. Herein, we show that intracerebroventricular injection of Aβ1-42 in mice (Aβ-mice) enhanced hippocampal Ras activation and expression, while 60 min incubation of hippocampal slices in Aβ1-42 (Aβ-slices) only elevated Ras activity. Aβ-mice showed deficits in spatial cognition and NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) in hippocampal CA1, but basal synaptic transmission was enhanced. The above effects of Aβ1-42 were corrected by the Ras inhibitor farnesylthiosalicylic acid (FTS). ERK2 phosphorylation increased, and Src phosphorylation decreased in Aβ-mice and Aβ1-42-slices. Both were corrected by FTS. In CA1 pyramidal cells of Aβ1-42-slices, the response of AMPA receptor and phosphorylation of GluR1 were enhanced with dependence on Ras activation rather than ERK signaling. In contrast, NMDA receptor (NMDAR) function and GluN2A/2B phosphorylation were downregulated in Aβ1-42-slices, which was recovered by application of FTS or the Src activator ouabain, and mimicked in control slices treated with the Src inhibitor PP2. The administration of PP2 impaired the spatial cognition and LTP induction in control mice and FTS-treated Aβ-mice. The treatment of Aβ-mice with ouabain rescued Aβ-impaired spatial cognition and LTP. Overall, the results indicate that the oligomeric Aβ1-42 hyperactivates Ras and thereby causes the downregulation of Src which impedes NMDAR-dependent LTP induction resulting in cognitive deficits.

Exosomes are cell-derived nanovesicles, and lately, cancer-derived exosomes have been reported to carry KRAS protein, which contributes to the malignancy of many cancers. In this study, farnesylthiosalicylic acid (FTS) was used to inhibit the activities of mutated KRAS in colon cancer SW480 cells to discover the potential link between KRAS activities and cancer-derived exosomes. We observed that FTS inhibits KRAS activity in SW480 cells, but promotes their exosome production. When the exosomal proteins of SW480 cells were profiled, a total of 435 proteins were identified with 16 of them showing significant changes (greater than or equal to two-fold) in response to FTS treatment. Protein network analysis suggests KRAS inhibition may trigger stress in the cells. In addition, a high level of acetyl-coA synthetase family member 4 protein which plays an important role in colon cancer survival was identified in the exosomes secreted by FTS-treated SW480 cells. The uptake of these exosomes suppresses the growth of some cell types, but in general exosomes from FTS-treated cells enhance the recipient cell survival when compared to that of untreated cells. Together our findings suggest that FTS may trigger stress in SW480 cells, and induce more exosomes secretion as the survival messenger to mitigate the impact of KRAS inhibition in colon cancer cells.

The Ras family of GTPases plays an important role in signaling nodes downstream to T cell receptor and CD28 activation, potentially lowering the threshold for T-cell receptor activation by autoantigens. Somatic mutation in NRAS or KRAS may cause a rare autoimmune disorder coupled with abnormal expansion of lymphocytes. T cells from rheumatoid arthritis (RA) patients show excessive activation of Ras/MEK/ERK pathway. The small molecule farnesylthiosalicylic acid (FTS) interferes with the interaction between Ras GTPases and their prenyl-binding chaperones to inhibit proper plasma membrane localization. In the present study, we tested the therapeutic and immunomodulatory effects of FTS and its derivative 5-fluoro-FTS (F-FTS) in the rat adjuvant-induced arthritis model (AIA). We show that AIA severity was significantly reduced by oral FTS and F-FTS treatment compared to vehicle control treatment. FTS was as effective as the mainstay anti-rheumatic drug methotrexate, and combining the two drugs significantly increased efficacy compared to each drug alone. We also discovered that FTS therapy inhibited both the CFA-driven in vivo induction of Th17 and IL-17/IFN-γ producing “double positive” as well as the upregulation of serum levels of the Th17-associated cytokines IL-17A and IL-22. By gene microarray analysis of effector CD4+ T cells from CFA-immunized rats, re-stimulated in vitro with the mycobacterium tuberculosis heat-shock protein 65 (Bhsp65), we determined that FTS abrogated the Bhsp65-induced transcription of a large list of genes (e.g., Il17a/f, Il22, Ifng, Csf2, Lta, and Il1a). The functional enrichment bioinformatics analysis showed significant overlap with predefined gene sets related to inflammation, immune system processes and autoimmunity. In conclusion, FTS and F-FTS display broad immunomodulatory effects in AIA with inhibition of the Th17-type response to a dominant arthritogenic antigen. Hence, targeting Ras signal-transduction cascade is a potential novel therapeutic approach for RA.

The Ras family of GTPases plays an important role in signaling nodes downstream to T cell receptor and CD28 activation, potentially lowering the threshold for T-cell receptor activation by autoantigens. Somatic mutation in NRAS or KRAS may cause a rare autoimmune disorder coupled with abnormal expansion of lymphocytes. T cells from rheumatoid arthritis (RA) patients show excessive activation of Ras/MEK/ERK pathway. The small molecule farnesylthiosalicylic acid (FTS) interferes with the interaction between Ras GTPases and their prenyl-binding chaperones to inhibit proper plasma membrane localization. In the present study, we tested the therapeutic and immunomodulatory effects of FTS and its derivative 5-fluoro-FTS (F-FTS) in the rat adjuvant-induced arthritis model (AIA). We show that AIA severity was significantly reduced by oral FTS and F-FTS treatment compared to vehicle control treatment. FTS was as effective as the mainstay anti-rheumatic drug methotrexate, and combining the two drugs significantly increased efficacy compared to each drug alone. We also discovered that FTS therapy inhibited both the CFA-driven in vivo induction of Th17 and IL-17/IFN-γ producing "double positive" as well as the upregulation of serum levels of the Th17-associated cytokines IL-17A and IL-22. By gene microarray analysis of effector CD4+ T cells from CFA-immunized rats, re-stimulated in vitro with the mycobacterium tuberculosis heat-shock protein 65 (Bhsp65), we determined that FTS abrogated the Bhsp65-induced transcription of a large list of genes (e.g., Il17a/f, Il22, Ifng, Csf2, Lta, and Il1a). The functional enrichment bioinformatics analysis showed significant overlap with predefined gene sets related to inflammation, immune system processes and autoimmunity. In conclusion, FTS and F-FTS display broad immunomodulatory effects in AIA with inhibition of the Th17-type response to a dominant arthritogenic antigen. Hence, targeting Ras signal-transduction cascade is a potential novel therapeutic approach for RA. [ABSTRACT FROM AUTHOR]

Primary glioblastomas (GBMs) commonly overexpress the oncogene epidermal growth factor receptor (EGFR), which leads to increased Ras activity. FTA, a novel Ras inhibitor, produced both time- and dose-dependent caspase-mediated apoptosis in GBM cell lines. EGFR-mediated increase in 3H-thymidine uptake was inhibited by FTA. FACS analysis was performed to determine the percent of apoptotic cells. The sub-Go population of GBM cells was increased from 4.5 to 13.8% (control) to over 45–53.6% in FTA-treated cells within 24 h. Furthermore, FTA also increased the activities of both caspase-3 and -9, and PARP cleavage. Treatment of GBMs with FTA before or after EGF addition to the cultures blocked phosphorylation of Akt and mitogen-activated protein kinases (MAPK). FTA also significantly reduced the amount of EGF-induced Ras-GTP as reflected by a decrease in the level of Ras bound to Raf-RBD-GST. This study demonstrates that inhibition of Ras methylation may provide a therapeutic target for the treatment of GBMs overexpressing EGFR.Cell Death and Differentiation (2006) 13, 642–651. doi:10.1038/sj.cdd.4401783; published online 21 October 2005 [ABSTRACT FROM AUTHOR]

The inhibition of human mesangial cell proliferation by S- trans, trans-farnesylthiosalicylic acid. Background. Many of the proliferative cytokines implicated in human mesangial cell (HMC) proliferation signal through the superfamily of Ras GTPases. The Ras antagonist, S- trans, trans- farnesylthiosalicylic acid (FTS), was used to investigate the effects of the inhibition of Ras signaling on HMC proliferation. Methods. Ras expression and membrane localization, MAPK, and Akt activation were analyzed by Western blotting. Ras activation was determined with a pull-down assay using the Ras-binding domain of Raf. HMC growth curves were assessed using the MTS assay of viable cell number, while DNA synthesis was measured with BrdU incorporation. Hoechst 33342 staining was used to determine apoptosis. Results. FTS reduced the membrane localization of Ras in both serum and platelet-derived growth factor (PDGF). FTS (7.5–20 μmol/L) potently inhibited PDGF-induced HMC proliferation but had no effect on serum-induced proliferation. FTS (10–20 μmol/L) inhibited both Ras and phospho-MAPK activation by serum and PDGF. Furthermore, FTS (10–20 μmol/L) increased HMC apoptosis in the presence of PDGF but not in serum. Moreover, PDGF-stimulated activation of the survival protein Akt was inhibited by FTS. In contrast, serum-stimulated activation of Akt was unaffected by FTS. Conclusion. FTS (5–20 μmol/L) inhibits PDGF-induced but not serum-induced HMC proliferation. FTS (10–20 μmol/L) also promotes HMC apoptosis in the presence of PDGF but not serum. These effects appear to be mediated by inhibitory effects on Ras-dependent signaling that occur as a result of the dislodgment of Ras from its membrane-anchorage sites by FTS. The selectivity of FTS toward PDGF-driven HMC proliferation suggests that FTS may be a valuable therapeutic in mesangioproliferative renal disease. [ABSTRACT FROM AUTHOR]

Cancer vaccines that make use of tumor antigens represent a promising therapeutic strategy by stimulating immune responses against tumors to generate long-term anti-tumor immunity. However, vaccines have shown limited clinical efficacy due to inefficient delivery. In this study, we focus on vaccine delivery assisted by nanocomplexes for cancer immunotherapy. Nanocomplex-mediated vaccination can efficiently deliver nucleic acids encoding neoantigens to lymphoid tissues and antigen-presenting cells. Polyethylenimine (PEI) was conjugated with farnesylthiosalicylic acid (FTS) to form micelles. Subsequent interaction with nucleic acids led to formation of polymer/nucleic acid nanocomplexes of well-controlled structure. Tumor transfection via FTS-PEI was much more effective than that by PEI, other PEI derivatives, or naked DNA. Significant numbers of transfected cells were also observed in draining lymph nodes (LNs). In vivo delivery of ovalbumin (OVA; a model antigen) expression plasmid (pOVA) by FTS-PEI led to a significant growth inhibition of the OVA-expressing B16 tumor through presentation of OVA epitopes as well as other epitopes via epitope spreading. Moreover, in vivo delivery of an endogenous melanoma neoantigen tyrosinase-related protein 2 (Trp2) also led to substantial tumor growth inhibition. FTS-PEI represents a promising transfection agent for effective gene delivery to tumors and LNs to mediate effective neoantigen vaccination., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Exosomes are cell-derived nanovesicles, and lately, cancer-derived exosomes have been reported to carry KRAS protein, which contributes to the malignancy of many cancers. In this study, farnesylthiosalicylic acid (FTS) was used to inhibit the activities of mutated KRAS in colon cancer SW480 cells to discover the potential link between KRAS activities and cancer-derived exosomes. We observed that FTS inhibits KRAS activity in SW480 cells, but promotes their exosome production. When the exosomal proteins of SW480 cells were profiled, a total of 435 proteins were identified with 16 of them showing significant changes (greater than or equal to two-fold) in response to FTS treatment. Protein network analysis suggests KRAS inhibition may trigger stress in the cells. In addition, a high level of acetyl-coA synthetase family member 4 protein which plays an important role in colon cancer survival was identified in the exosomes secreted by FTS-treated SW480 cells. The uptake of these exosomes suppresses the growth of some cell types, but in general exosomes from FTS-treated cells enhance the recipient cell survival when compared to that of untreated cells. Together our findings suggest that FTS may trigger stress in SW480 cells, and induce more exosomes secretion as the survival messenger to mitigate the impact of KRAS inhibition in colon cancer cells. [ABSTRACT FROM AUTHOR]

The activity of Ras, a small GTPase protein, is increased in brains with Alzheimer's disease. The objective of this study was to determine the influence of oligomeric Aβ1-42 on the activation of Ras, and the involvement of the Ras hyperactivity in Aβ1-42-induced deficits in spatial cognition and hippocampal synaptic plasticity. Herein, we show that intracerebroventricular injection of Aβ1-42 in mice (Aβ-mice) enhanced hippocampal Ras activation and expression, while 60 min incubation of hippocampal slices in Aβ1-42 (Aβ-slices) only elevated Ras activity. Aβ-mice showed deficits in spatial cognition and NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) in hippocampal CA1, but basal synaptic transmission was enhanced. The above effects of Aβ1-42 were corrected by the Ras inhibitor farnesylthiosalicylic acid (FTS). ERK2 phosphorylation increased, and Src phosphorylation decreased in Aβ-mice and Aβ1-42-slices. Both were corrected by FTS. In CA1 pyramidal cells of Aβ1-42-slices, the response of AMPA receptor and phosphorylation of GluR1 were enhanced with dependence on Ras activation rather than ERK signaling. In contrast, NMDA receptor (NMDAR) function and GluN2A/2B phosphorylation were downregulated in Aβ1-42-slices, which was recovered by application of FTS or the Src activator ouabain, and mimicked in control slices treated with the Src inhibitor PP2. The administration of PP2 impaired the spatial cognition and LTP induction in control mice and FTS-treated Aβ-mice. The treatment of Aβ-mice with ouabain rescued Aβ-impaired spatial cognition and LTP. Overall, the results indicate that the oligomeric Aβ1-42 hyperactivates Ras and thereby causes the downregulation of Src which impedes NMDAR-dependent LTP induction resulting in cognitive deficits. [ABSTRACT FROM AUTHOR]