Exosomes secreted by Fusobacterium nucleatum-infected colon cancer cells transmit resistance to oxaliplatin and 5-FU by delivering hsa_circ_0004085.

BACKGROUND: A large number of Fusobacterium nucleatum (Fn) are present in colorectal cancer (CRC) tissues of patients who relapse after chemotherapy, and Fn has been reported to promote oxaliplatin and 5-FU chemoresistance in CRC. Pathogens such as bacteria and parasites stimulate exosome production in tumor cells, and the regulatory mechanism of exosomal circRNA in the transmission of oxaliplatin and 5-FU chemotherapy resistance in Fn-infected CRC remains unclear. METHODS: Hsa_circ_0004085 was screened by second-generation sequencing of CRC tissues. The correlation between hsa_circ_0004085 and patient clinical response to oxaliplatin/5-FU was analyzed. Exosome tracing experiments and live imaging systems were used to test the effect of Fn infection in CRC on the distribution of hsa_circ_0004085. Colony formation, ER tracking analysis and immunofluorescence were carried out to verify the regulatory effect of exosomes produced by Fn-infected CRC cells on chemotherapeutic resistance and ER stress. RNA pulldown, LC-MS/MS analysis and RIP were used to explore the regulatory mechanism of downstream target genes by hsa_circ_0004085. RESULTS: First, we screened out hsa_circ_0004085 with abnormally high expression in CRC clinical samples infected with Fn and found that patients with high expression of hsa_circ_0004085 in plasma had a poor clinical response to oxaliplatin/5-FU. Subsequently, the circular structure of hsa_circ_0004085 was identified. Fn infection promoted hsa_circ_0004085 formation by hnRNP L and packaged hsa_circ_0004085 into exosomes by hnRNP A1. Exosomes produced by Fn-infected CRC cells transferred hsa_circ_0004085 between cells and delivered oxaliplatin/5-FU resistance to recipient cells by relieving ER stress. Hsa_circ_0004085 enhanced the stability of GRP78 mRNA by binding to RRBP1 and promoted the nuclear translocation of ATF6p50 to relieve ER stress. CONCLUSIONS: Plasma levels of hsa_circ_0004085 are increased in colon cancer patients with intracellular Fn and are associated with a poor response to oxaliplatin/5-FU. Fn infection promoted hsa_circ_0004085 formation by hnRNP L and packaged hsa_circ_0004085 into exosomes by hnRNP A1. Exosomes secreted by Fn-infected CRC cells deliver hsa_circ_0004085 between cells. Hsa_circ_0004085 relieves ER stress in recipient cells by regulating GRP78 and ATF6p50, thereby delivering resistance to oxaliplatin and 5-FU.

Exosome-transmitted FOSL1 from cancer-associated fibroblasts drives colorectal cancer stemness and chemo-resistance through transcriptionally activating ITGB4.

Cancer-associated fibroblasts (CAFs) have been proved to facilitate colorectal cancer (CRC) development, either with boosting chemo-resistance by communicating with CRC cells in the tumor microenvironment. However, the underlying molecular mechanisms remain largely unclear. Relative expressions of FOSL1 and ITGB4, either with their correlations in CRC tissues, were assessed using qRT-PCR analysis. Also, Kaplan-Meier survival analysis was employed for evaluating the prognosis. Identification of CAFs was determined by the detection of specific makers (α-SMA, FAP, and FSP1) using western blot and immunofluorescence staining. Cell proliferation, self-renewal capacity, and cell apoptosis were estimated by CCK-8, sphere-formation, and flow cytometry assays. Transcriptional regulation of FOSL1 on integrin ß4 (ITGB4) was confirmed using ChIP and dual-luciferase reporter assays. Increased FOSL1 and ITGB4 in CRC tissues were both positively correlated with the poor prognosis of CRC patients. Interestingly, FOSL1 was enriched in the CAFs isolated from CRC stroma, instead of ITGB4. CRC cells under a co-culture system with CAFs-conditioned medium (CAFs-CM) exhibited increased FOSL1, promotive cell proliferation, and reduced apoptosis, while these effects could be blocked by exosome inhibitor (GW4869). Moreover, CAFs-derived exosomal FOSL1 was validated to enhance proliferative ability and oxaliplatin resistance of CRC cells. Our results uncovered that CAFs-derived exosomes could transfer FOSL1 to CRC cells, thereby promoting CRC cell proliferation, stemness, and oxaliplatin resistance by transcriptionally activating ITGB4.

MAPK-ERK-CREB signaling pathway upregulates Nav1.6 in oxaliplatin-induced neuropathic pain in the rat.

The voltage-gated sodium channel subtype Nav1.6 is involved in the electrophysiological changes of primary sensory neurons that occur in oxaliplatin-induced neuropathic pain, but its regulatory mechanism remains unclear. In this study, Western blot, RT-qPCR, immunofluorescence staining, chromatin immunoprecipitation were used to prove the mechanism of MAPK-ERK-CREB signaling pathway participating in oxaliplatin-induced neuropathic pain by regulating Nav1.6. The results showed that p-Raf1 and p-ERK, key molecules in MAPK/ERK pathway, and Nav1.6 were significantly increased in DRGs of oxaliplatin-induced neuropathic pain rats. Inhibition of p-Raf1 and p-ERK respectively not only reduced the expression of Nav1.6 protein in DRGs of OXA rats, but also caused a decrease in Nav1.6 mRNA, which led us to further explore the transcription factor CREB regulated by MAPK/ERK pathway. Results showed that CREB was co-distributed with Nav1.6. Inhibition of CREB resulted in decreased mRNA and protein expression of Nav1.6, and alleviated oxaliplatin-induced neuropathic pain. A chromatin immunoprecipitation experiment proved that OXA caused p-CREB to directly bind to the promoter region of Scn8A, which is the encoding gene for Nav1.6, and promote the transcription of Scn8A. In summary, in this study, we found that oxaliplatin can activate the MAPK/ERK pathway, which promotes the expression and activation of CREB and leads to an increase in Scn8A transcription, and then leads to an increase in Nav1.6 protein expression to enhance neuronal excitability and cause pain. This study provides an experimental basis for the molecular mechanism of sodium channel regulation in oxaliplatin-induced neuropathic pain.

Satellite glial cell-secreted exosomes after in-vitro oxaliplatin treatment presents a pro-nociceptive effect for dorsal root ganglion neurons and induce mechanical hypersensitivity in naïve mice.

BACKGROUND: The pathophysiological mechanism underlying chemotherapy-induced neuropathic pain (CINP) remains unclear. Sensory neuronal hypersensitivity in the dorsal root ganglion (DRG) is essential for the onset and maintenance of chronic pain. Satellite glial cells (SGCs) in the DRG potentially affect the function of sensory neurons, possibly by mediating extracellular or paracrine signaling. Exosomes play an essential role in cell-cell communication. However, the role of SGC-secreted exosomes in glia-neuron communication and CINP remains unclear. METHODS: SGCs and sensory neurons were cultured from the DRG of mice. The SGCs were treated with 4 µM oxaliplatin for 24 h. Glial fibrillary acid protein (GFAP) and connexin-43 (Cx-43) expressions in the SGCs were examined with immunocytochemistry (ICC). Enzyme-linked immunosorbent assay (ELISA) detected cytokine release in the SGCs after oxaliplatin treatment. Subsequently, SGC-secreted exosomes were collected using ultracentrifugation and identified by nanoparticle tracking analysis, transmission electron microscopy, and western blotting. Subsequently, DRG neurons were incubated with SGC-secreted exosomes for 24 h. The percentage of reactive oxygen species (ROS)-positive neurons was detected using flow cytometry, and acid-sensing ion channel 3 (ASIC3) and transient receptor potential vanilloid 1 (TRPV1) expression were examined by western blotting. SGC-secreted exosomes were intrathecally injected into naïve mice. The mechanical withdrawal threshold was assessed 24, 48, and 72 h following the injection. TRPV1 expression in the DRG was examined 72 h after intrathecal injection. Furthermore, differentially expressed (DE) miRNAs within the SGC-secreted exosomes were detected using RNA sequencing and bioinformatics analysis. Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses were performed to predict the function of the target genes of DE miRNAs. Finally, the DE miRNAs with pain regulation potential were identified in silico. RESULTS: After in-vitro oxaliplatin treatment, ICC showed an increase in the immunoreactivity of GFAP and Cx-43 in the SGCs. ELISA results suggested an increased release of tumor necrosis factor-α and interleukin (IL)-1ß, but a decreased release of IL-10. Oxaliplatin treatment increased the secretion of exosomes in the SGCs from 4.34 to 5.99 × 1011 (particles/ml). The exosome-specific markers CD9 and TSG101 were positive, whereas calnexin was negative for the obtained exosomes. Additionally, the SGC-secreted exosomes were endocytosed by DRG neurons after co-incubation. Moreover, after incubation with conditioned SGC-secreted exosomes (after 4 µM oxaliplatin treatment), the percentage of ROS-positive DRG neurons increased and ASIC3 and TRPV1 expressions were upregulated. After the intrathecal injection of the conditioned SGC-secreted exosomes, the mice presented with mechanical hypersensitivity and TRPV1 expression upregulation in the DRG. Notably, 25 and 120 significantly upregulated and downregulated miRNAs, respectively, were identified in the conditioned SGC-secreted exosomes. When predicting the function of target genes of DE miRNAs, certain GO terms, such as synapse organization, neurogenesis regulation, histone modification, and pain-related KEGG or Reactome pathways, including vascular endothelial growth factor A-vascular endothelial growth factor receptor 2, mammalian target of rapamycin, and mitogen-activated protein kinase signaling pathways, related to nervous system function were predicted. Finally, 27 pain regulation-related miRNAs, including miR-324-3p, miR-181a-5p, and miR-122-5p, were identified in silico. CONCLUSION: Our study demonstrates that SGC-secreted exosomes after in-vitro oxaliplatin treatment present a pro-nociceptive effect for DRG neurons and induce mechanical hypersensitivity in naïve mice, possibly via the contained miRNA cargo. Identifying the candidate miRNAs and verifying their functions in vivo are required to elucidate the exosomes mediating 'glia-neuron' communication under CINP condition.

Nose to brain delivery of naringin-loaded chitosan nanoparticles for potential use in oxaliplatin-induced chemobrain in rats: impact on oxidative stress, cGAS/STING and HMGB1/RAGE/TLR2/MYD88 inflammatory axes.

OBJECTIVES: Oxaliplatin induces chemobrain in cancer patients/survivors. Nutraceutical naringin has antioxidant and anti-inflammatory properties with low oral bioavailability. Our aim was to formulate naringin in chitosan nanoparticles for nose to brain delivery and assess its neuroprotective effect against oxaliplatin-induced chemobrain in rats. METHODS: Naringin chitosan nanoparticles were prepared by ionic gelation. Rats were administered oral naringin (80 mg/kg), intranasal naringin (0.3 mg/kg) or intranasal naringin-loaded chitosan nanoparticles (0.3 mg/kg). Naringin's neuroprotective efficacy was assessed based on behavioral tests, histopathology, and measuring oxidative stress and inflammatory markers. RESULTS: Selected nanoparticles formulation showed drug loading of 5%, size of 150 nm and were cationic. Intranasal naringin administration enhanced memory function, inhibited hippocampal acetylcholinesterase activity, and corrected oxaliplatin-induced histological changes. Moreover, it reduced malondialdehyde and elevated reduced glutathione hippocampal levels. Furthermore, it decreased levels of inflammatory markers: NF-kB and TNF-α by 1.25-fold. Upstream to this inflammatory status, intranasal naringin downregulated the hippocampal protein levels of two pathways: cGAS/STING and HMGB1/RAGE/TLR2/MYD88. CONCLUSION: Intranasal naringin-loaded chitosan nanoparticles showed superior amelioration of oxaliplatin-induced chemobrain in rats at a dose 267-fold lower to that administered orally. The potential involvement of cGAS/STING and HMGB1/RAGE/TLR2/MYD88 pathways in the mechanistic process of either oxaliplatin-induced chemobrain or naringin-mediated neuroprotection was evidenced.

Reduction of SIRT1-Mediated Epigenetic Upregulation of Nav1.7 Contributes to Oxaliplatin-Induced Neuropathic Pain.

BACKGROUND: Clinically, neuropathic pain is a severe side effect of oxaliplatin chemotherapy, which usually leads to dose reduction or cessation of treatment. Due to the unawareness of detailed mechanisms of oxaliplatin-induced neuropathic pain, it is difficult to develop an effective therapy and limits its clinical use. OBJECTIVES: The aim of the present study was to identify the role of sirtuin 1 (SIRT1) reduction in epigenetic regulation of the expression of voltage-gated sodium channels 1.7 (Nav1.7) in the dorsal root ganglion (DRG) during oxaliplatin-induced neuropathic pain. STUDY DESIGN: Controlled animal study. SETTING: University laboratory. METHODS: The von Frey test was performed to evaluate pain behavior in rats. Real-time quantitative polymerase chain reaction, western blotting, electrophysiological recording, chromatin immunoprecipitation, and small interfering RNA (siRNA) were used to illustrate the mechanisms. RESULTS: In the present study, we found that both the activity and expression of SIRT1 were significantly decreased in rat DRG following oxaliplatin treatment. The activator of SIRT1, resveratrol, not only increased the activity and expression of SIRT1, but also attenuated the mechanical allodynia following oxaliplatin treatment. In addition, local knockdown of SIRT1 by intrathecal injection of SIRT1 siRNA caused mechanical allodynia in naive rats. Besides, oxaliplatin treatment enhanced the action potential firing frequency of DRG neurons and the expression of Nav1.7 in DRG and activation of SIRT1 by resveratrol reversed this effect. Furthermore, blocking Nav1.7 by ProTx II (a selective Nav1.7 channel blocker) reversed oxaliplatin-induced mechanical allodynia. In addition, histone H3 hyperacetylation at the Nav1.7 promoter in DRG of rats following oxaliplatin treatment was significantly suppressed by activation of SIRT1 with resveratrol. Moreover, both the expression of Nav1.7 and histone H3 acetylation at the Nav1.7 promoter were upregulated in the DRG by local knockdown of SIRT1 with SIRT1 siRNA in naive rats. LIMITATIONS: More underlying mechanism(s) of SIRT1 reduction after oxaliplatin treatment needs to be explored in future research. CONCLUSIONS: These findings suggest that reduction of SIRT1-mediated epigenetic upregulation of Nav1.7 in the DRG contributes to the development of oxaliplatin-induced neuropathic pain in rats. The intrathecal drug delivery treatment of activating SIRT1 might be a novel therapeutic option for oxaliplatin-induced neuropathic pain.

siRNA-E6 sensitizes HPV-16-related cervical cancer through Oxaliplatin: an in vitro study on anti-cancer combination therapy.

BACKGROUND: Persistent infection with high-risk Human papillomaviruses (HPV), such as hr-HPV-16 and hr-HPV-18, lead to cervical cancer, the fourth most common cancer in the world. In the present study, we investigated the alteration of E6 oncogene expression by E6-specific short interfering RNA (siRNA) combined with Oxaliplatin. METHODS: The cervical cancer cell line, CaSki, was transfected with E6-siRNA, then treated with Oxaliplatin. The cellular genes, such as p53, MMP9, Nanog, and caspases expression, were assessed by quantitative real-time PCR. The cell death rate, cell cycle, and cell viability were assessed by Annexin V/PI staining, DAPI staining, and MTT test, respectively. Furthermore, colony formation assay and scratch test determined the stemness ability and cell metastasis, respectively. RESULTS: Combination therapy increased the re-expression of genes involved in the p53-dependent apoptosis pathway (increase in apoptosis to 44.2%), and reduced stemness and metastasis ability compared to either siRNA or Oxaliplatin monotherapy. Together, our results demonstrate that E6-siRNA and Oxaliplatin combination increased the cervical cancer cells' sensitivity to Oxaliplatin and decreased the survival rate, proliferation, and metastasis, and consequently escalated apoptosis rate, induced cell cycle arrest in the sub-G1 stage, and reduced the chemotherapy drug dosage. CONCLUSION: Inhibition of E6 oncogene expression and subsequent E6-siRNA with Oxaliplatin combination therapy could be a novel strategy for cervical cancer treatment.

Calcipotriol abrogates cancer-associated fibroblast-derived IL-8-mediated oxaliplatin resistance in gastric cancer cells via blocking PI3K/Akt signaling.

Activation of vitamin D receptor (VDR) in cancer-associated fibroblasts (CAFs) has been implicated in hesitating tumor progression and chemoresistance of several human malignancies. Yet, the role of VDR in CAF-induced chemotherapy resistance of gastric cancer (GC) cells remains elusive. In this study we first conducted immunohistochemistry analysis on tissue microarrays including 88 pairs of GC and normal mucosa samples, and provided clinical evidence that VDR was mainly expressed in gastric mucous cells but almost invisible in CAFs, and VDR expression was negatively correlated with malignant clinical phenotype and advanced stages, low VDR expression confers to poor overall survival rate of patients with GC. In a co-culture system of primary CAFs and cancer cells, we showed that treatment of HGC-27 and AGS GC cells with VDR ligand calcipotriol (Cal, 500 nM) significantly inhibited CAF-induced oxaliplatin resistance. By using RNA-sequencing and Human Cytokine Antibody Array, we demonstrated that IL-8 secretion from CAFs induced oxaliplatin resistance via activating the PI3K/AKT pathway in GC, whereas Cal treatment greatly attenuated the tumor-supportive effect of CAF-derived IL-8 on GC cells. Taken together, this study verifies the specific localization of VDR in GC tissues and demonstrates that activation of VDR abrogates CAF-derived IL-8-mediated oxaliplatin resistance in GC via blocking PI3K/Akt signaling, suggesting vitamin D supplementation as a potential strategy of enhancing the anti-tumor effect of chemotherapy in GC.

Comparison of the immunomodulatory potential of platinum-based anti-cancer drugs and anthracyclins on human monocyte-derived cells.

Macrophages and dendritic cells (DCs) are important contributors to anti-tumor immune responses. However, these highly plastic cells are also the primary targets of tumor manipulation, which may result in the development of tumor-promoting subtypes. The effect of chemotherapeutic agents on tumor cells is an area of intense study, but little is known about their effects on innate immune cells.We investigated the effects of four chemotherapeutic drugs (two platinum-based agents; oxaliplatin and cisplatin, and two anthracyclines; doxorubicin and epirubicin) on the differentiation, function, and viability of macrophages and DCs. Macrophages and DCs were differentiated from monocytes in the presence of these chemotherapeutic drugs and we compared their cell surface receptor expression, cytokine production, and chemotactic- and T-cell-polarizing ability.We have shown that differentiation in the presence of anthracyclines dose-dependently increases CTLA-4 expression in DCs. Antineoplastic agent-driven differentiation strongly modified the CCL2- or CCL5-induced chemotactic activity of both macrophages and DCs. DCs differentiated in the presence of high-dose cisplatin and a low dose of epirubicin promoted regulatory T-cell development, whereas oxaliplatin at specific doses induced both DCs and macrophages to enhance cytotoxic T-cell responses. Furthermore, we found that inflammatory macrophages are more sensitive to doxorubicin-induced cell death than their counterparts.In summary, our results confirm that chemotherapeutic agents acting on a similar basis may have different effects on the anti-tumor immune response. Treatment with optimal dose, combinations, and timing of chemotherapy may determine tumor immunity and the metastatic potential of tumors.

SNAI1-expressing fibroblasts and derived-extracellular matrix as mediators of drug resistance in colorectal cancer patients.

Resistance to antitumor treatments is one of the most important problems faced by clinicians in the management of colorectal cancer (CRC) patients. Cancer-Associated Fibroblasts (CAFs) are the main producers and remodelers of the extracellular matrix (ECM), which is directly involved in drug resistance mechanisms. Primary Normal Fibroblasts (NFs) and CAFs and cell lines (fibroblasts and tumor cells), were used to generate ECM and to identify its role in the oxaliplatin and cetuximab chemoresistance processes of CRC cells mediated by SNAI1-expressing fibroblasts. Matrices generated by Snai1 KO MEFs (Knockout Mouse Embryonic Fibroblasts) confer less resistance on oxaliplatin and cetuximab than wild-type MEF-derived matrices. Similarly, matrices derived from CAFs cause greater survival of colorectal cancer cells than NF-derived matrices, in a similar way to Snai1 expression levels. In addition, Snail1 expression in fibroblasts regulates drug resistance and metabolism gene expression in tumor cells mediated by ECM. Finally, a series of 531 patients (TCGA) with CRC was used to assess the role of SNAI1 expression in patients' prognosis indicating an association between tumor SNAI1 expression and overall survival in colon cancer patients but not in rectal cancer patients. SNAI1 expression in CRC cancer patients, together with in vitro experimentation, suggests the possible use of SNAI1 expression in tumor-associated fibroblasts as a predictive biomarker of response to oxaliplatin and cetuximab treatments in patients with CRC.

In vitro exosomal transfer of Nrf2 led to the oxaliplatin resistance in human colorectal cancer LS174T cells.

Chemotherapy resistance is a serious pitfall in the treatment of colon cancers (CCs). Previous studies have found that exosomes (Exo) play a pivotal role in tumor drug resistance via the transfer of proteins and genetic materials to the acceptor cells. To date, the mechanisms orchestrating Exo-derived resistance in cancer cells have been the center of attention. Herein, we aimed to evaluate the role of exosomal nuclear factor erythroid 2-related factor 2 (Nrf2) on oxaliplatin (1-OHP) resistance in human colorectal cancer LS174T cells in vitro. To this end, exosomal-Nrf2-mediated 1-OHP resistance was examined using different assays. Exo was isolated from resistant LS174T cells (LS174T/R) and added to the culture medium of sensitive LS174T cells (LS174T/S). According to our data, LS174T/S cells successfully adsorbed PKH26-Exo driven from LS174T/R cells. Western blotting showed an increased Nrf2 level in Exo isolated from LS174T/R cells compared to LS174T/S cell-derived Exo (p < .05). The incubation of LS174T/S cells with LS174T/R-derived Exo increased half-maximal inhibitory concentration values in response to treatment with 1-OHP (p < .05). Besides this, the apoptotic changes were diminished in LS174T/S cells after incubation with LS174T/R-derived Exo. Of note, the exposure of LS174T/S cells to LS174T/R cell-derived Exo increased the expression of Nrf2 and P-glycoprotein (P-gp) compared to the nontreated LS174T/S cells (p < .05). In line with these changes, lower intracellular Rhodamin 123 content was detected in Exo-treated cells compared to the nontreated LS174T/S cells. Exo increased migration and clonogenic capacity of LS174T/S cells after incubation with Exo-derived from resistant cells. Of note, inhibition of Nrf2 with a specific blocker, brusatol, blunted these effects. Taken together, Exo-mediated transfer of Nrf2 is involved in the development of oxaliplatin resistance in CC cells by upregulating P-gp.

Biomimetic nanoparticles blocking autophagy for enhanced chemotherapy and metastasis inhibition via reversing focal adhesion disassembly.

BACKGROUND: Autophagy is a conserved catabolic process, which plays an important role in regulating tumor cell motility and degrading protein aggregates. Chemotherapy-induced autophagy may lead to tumor distant metastasis and even chemo-insensitivity in the therapy of hepatocellular carcinoma (HCC). Therefore, a vast majority of HCC cases do not produce a significant response to monotherapy with autophagy inhibitors. RESULTS: In this work, we developed a biomimetic nanoformulation (TH-NP) co-encapsulating Oxaliplatin (OXA)/hydroxychloroquine (HCQ, an autophagy inhibitor) to execute targeted autophagy inhibition, reduce tumor cell migration and invasion in vitro and attenuate metastasis in vivo. The tumor cell-specific ligand TRAIL was bioengineered to be stably expressed on HUVECs and the resultant membrane vesicles were wrapped on OXA/HCQ-loaded PLGA nanocores. Especially, TH-NPs could significantly improve OXA and HCQ effective concentration by approximately 21 and 13 times in tumor tissues compared to the free mixture of HCQ/OXA. Moreover, the tumor-targeting TH-NPs released HCQ alkalized the acidic lysosomes and inhibited the fusion of autophagosomes and lysosomes, leading to effective blockade of autophagic flux. In short, the system largely improved chemotherapeutic performance of OXA on subcutaneous and orthotopic HCC mice models. Importantly, TH-NPs also exhibited the most effective inhibition of tumor metastasis in orthotopic HCCLM3 models, and in the HepG2, Huh-7 or HCCLM3 metastatic mice models. Finally, we illustrated the enhanced metastasis inhibition was attributed to the blockade or reverse of the autophagy-mediated degradation of focal adhesions (FAs) including E-cadherin and paxillin. CONCLUSIONS: TH-NPs can perform an enhanced chemotherapy and antimetastatic effect, and may represent a promising strategy for HCC therapy in clinics.

[Elucidation for Intratumor Localization of a DDS-based Anticancer Drug and Enhancement of Its Therapeutic Effects via Improvement of the Tumor Microenvironment].

In the development of drug delivery system (DDS)-based anticancer drugs, the techniques for the intratumor mapping and quantification of active pharmaceutical ingredients (API) in pharmaceuticals must be pivotal for predicting pharmacological effects and adverse events. X-ray fluorescence spectrometry (XRF) is a potent analytical tool for mapping/quantifying platinum pharmaceutics such as oxaliplatin (l-OHP) and its liposomal formulation. In recent studies, we employed XRF to visualize the intratumor micro-distribution of l-OHP in a tumor-bearing model mouse intravenously injected with either free l-OHP or l-OHP liposomes. The intratumor distribution of l-OHP within tumor sections could be determined by XRF to detect platinum atoms. After treatment with the liposomal formulation, the l-OHP was localized near the tumor vessels and, via repeated injections, increasingly accumulated in tumors by a much greater degree than treatment with free l-OHP. The repeated injections of l-OHP liposomes improved the vascular permeability via inducing the apoptosis of tumor cells near the tumor vessels, which should improve the tumor microenvironment and enhance the intratumor accumulation of repeated doses of l-OHP liposomes. The proposed process was also used to visualize the intratumor distribution of l-OHP in rectal cancer specimens resected from a patient who had received l-OHP-based preoperative chemotherapy. We further revealed that neutralization of an acidic tumor microenvironment via oral administration with NaHCO3 could improve the therapeutic efficacy of weakly basic anticancer agent-encapsulating liposomes. Collectively, mapping/quantifying the intratumor API in DDS drugs and/or improving the tumor microenvironment would be an effective means to accelerate the clinical development of DDS-based anticancer drugs.

Rational Design of a Modality-Specific Inhibitor of TRPM8 Channel against Oxaliplatin-Induced Cold Allodynia.

Platinum-based compounds in chemotherapy such as oxaliplatin often induce peripheral neuropathy and neuropathic pain such as cold allodynia in patients. Transient Receptor Potential Melastatin 8 (TRPM8) ion channel is a nociceptor critically involved in such pathological processes. Direct blockade of TRPM8 exhibits significant analgesic effects but also incurs severe side effects such as hypothermia. To selectively target TRPM8 channels against cold allodynia, a cyclic peptide DeC-1.2 is de novo designed with the optimized hot-spot centric approach. DeC-1.2 modality specifically inhibited the ligand activation of TRPM8 but not the cold activation as measured in single-channel patch clamp recordings. It is further demonstrated that DeC-1.2 abolishes cold allodynia in oxaliplatin treated mice without altering body temperature, indicating DeC-1.2 has the potential for further development as a novel analgesic against oxaliplatin-induced neuropathic pain.

Delivery of oxaliplatin to colorectal cancer cells by folate-targeted UiO-66-NH2.

Oxaliplatin is being used in different malignancies and several side effects are reported for patients taking Oxaliplatin, including peripheral neuropathy, nausea and vomiting, diarrhea, mouth sores, low blood counts, fatigue, loss of appetite, etc. Here we have developed a targeted anticancer drug delivery system based on folate-conjugated amine-functionalized UiO-66 for the delivery of oxaliplatin (OX). UiO-66-NH2 (U) and UiO-66-NH2-FA(FU) were pre-functionalized by the incorporation of folic acid (FA) into the structure via coordination of the carboxylate group of FA. The FTIR spectra of drug-loaded U and FU showed the presence of new carboxylic and aliphatic groups of OX and FA. Powder X-ray diffraction (PXRD) patterns were matched accordingly with the reference pattern and FESEM results showed semi-spherical particles (115-128 nm). The evaluated amounts of OX in U and FU were calculated 304.5 and 293 mg/g, respectively. The initial burst release of OX was 15.7% per hour for U(OX) and 10.8% per hour for FU(OX). The final release plateau gives 62.9% and 52.3% for U(OX) and FU(OX). To evaluate the application of the prepared delivery platform, they were tested on colorectal cancer cells (CT-26) via MTT assay, cell migration assay, and spheroid model. IC50 values obtained from MTT assay were 21.38, 95.50, and 18.20 µg/mL for OX, U(OX), and FU(OX), respectively. After three days of treatment, the CT26 spheroids at two doses of 500 and 50 µg/mL of U(OX) and FU(OX) showed volume reduction. Moreover, the oxidative behavior of the prepared systems within the cell was assessed by total thiol, malondialdehyde, and superoxide dismutase activity. The results showed that FU(OX) had higher efficacy in preventing the growth of CT-26 spheroid, and was more effective than oxaliplation in cell migration inhibition, and induced higher oxidative stress and apoptosis.

Downregulation of metallothionein-2 contributes to oxaliplatin-induced neuropathic pain.

BACKGROUND: We previously reported a correlation between small doses of oxaliplatin penetrating onto the spinal cord and acute pain after chemotherapy. Here, we propose that MT2 within the spinal dorsal horns participates in the development of oxaliplatin-induced neuropathic pain and may be a pharmacological target for the prevention and treatment of chemotherapy-induced peripheral neuropathy (CIPN). METHODS: The rat model of CIPN was established by 5 consecutive injections of oxaliplatin (0.4 mg/100 g/day). Genetic restoration of neuron-specific metallothionein-2 was implemented 21 days before oxaliplatin treatment, and also, genetic inhibition by metallothionein-2 siRNA was performed. Mechanical allodynia and locomotor activity were assayed. Cell-specific expression of metallothionein-2, the mRNA levels of pro-inflammatory cytokines, nuclear translocation of NF-κB, the protein levels of expression of IκB-α, and interaction between IκB-α and P65 were evaluated in the spinal dorsal horns. Also, in vitro interaction of sequentially deleted IκB-α promoter with metallothionein-2 was used to assess the signal transduction mechanism. RESULTS: We found that oxaliplatin induced downregulation of metallothionein-2 in rat spinal cord neurons. By contrast, genetic restoration of metallothionein-2 in the spinal dorsal horn neuron blocked and reversed neuropathic pain in oxaliplatin-treated rats of both sexes, whereas genetic inhibition of metallothionein-2 triggered neuropathic pain in normal rats. Overall locomotor activity was not impaired after the genetic alterations of metallothionein-2. At the molecular level, metallothionein-2 modulated oxaliplatin-induced neuroinflammation, activation of NF-κB, and inactive transcriptional expression of IκB-α promoter, and these processes could be blocked by genetic restoration of metallothionein-2 in the spinal dorsal horn neurons. CONCLUSIONS: Metallothionein-2 is a potential target for the prevention and treatment of CIPN. A reduction of NF-κB activation and inflammatory responses by enhancing the transcription of IκB-α promoter is proposed in the mechanism.

Host-Guest Complexation of Oxaliplatin and Para-Sulfonatocalix[n]Arenes for Potential Use in Cancer Therapy.

P-sulfonatocalix[n]arenes have demonstrated a great potential for encapsulation of therapeutic drugs via host-guest complexation to improve solubility, stability, and bioavailability of encapsulated drugs. In this work, guest-host complexes of a third-generation anticancer drug (oxaliplatin) and p-4-sulfocalix[n]arenes (n = 4 and 6; p-SC4 and p-SC6, respectively) were prepared and investigated, using 1H NMR, UV, Job's plot analysis, and DFT calculations, for use as cancer therapeutics. The peak amplitude of the prepared host-guest complexes was linearly proportional to the concentration of oxaliplatin in the range of 1.0 × 10-5 M-1 to 2.1 × 10-4 M-1. The reaction stoichiometry between either p-SC4 or p-SC6 and oxaliplatin in the formed complexes was 1:1. The stability constants for the complexes were 5.07 × 104 M-1 and 6.3 × 104 M-1. These correspond to complexation free energy of -6.39 and -6.52 kcal/mol for p-SC4 and p-SC6, respectively. Complexation between oxaliplatin and p-SC4 or p-SC6 was found to involve hydrogen bonds. Both complexes exhibited enhanced biological and high cytotoxic activities against HT-29 colorectal cells and MCF-7 breast adenocarcinoma compared to free oxaliplatin, which warrants further investigation for cancer therapy.

Tumor-Activated Size-Enlargeable Bioinspired Lipoproteins Access Cancer Cells in Tumor to Elicit Anti-Tumor Immune Responses.

The limited lymphocytes infiltration and immunosuppression in tumor are the major challenges of cancer immunotherapy. The use of immunogenic cell death (ICD)-inducing agents has potential to potentiate antitumor immune responses, but is tremendously hampered by the poor delivery efficiency. Herein, a tumor-activated size-enlargeable bioinspired lipoprotein of oxaliplatin (TA-OBL) is designed to access cancer cells and boost the ICD-induced antitumor immunity for synergizing immune-checkpoint blockades (ICBs)-mediated immunotherapy. TA-OBL is constructed by integrating a legumain-sensitive melittin conjugate for improving intratumoral permeation and cancer cell accessibility, a pH-sensitive phospholipid for triggering size-enlargement and drug release in intracellular acidic environments, a nitroreductase-sensitive hydrophobic oxaliplatin prodrug (N-OXP) for eliciting antitumor immunity into the bioinspired nano-sized lipoprotein system. TA-OBL treatment produced robust antitumor immune responses and its combination with ICBs demonstrates strong therapeutic benefits with delayed tumor growth and extended survival rate, making it a promising delivery nanoplatform to elicit antitumor immunity for cancer immunotherapy.

Direct Binding of Cisplatin to p22phox, an Endoplasmic Reticulum (ER) Membrane Protein, Contributes to Cisplatin Resistance in Oral Squamous Cell Carcinoma (OSCC) Cells.

Prolonged treatment with cisplatin (CDDP) frequently develops chemoresistance. We have previously shown that p22phox, an endoplasmic reticulum (ER) membrane protein, confers CDDP resistance by blocking CDDP nuclear entry in oral squamous cell carcinoma (OSCC) cells; however, the underlying mechanism remains unresolved. Using a fluorescent dye-labeled CDDP, here we show that CDDP can bind to p22phox in both cell-based and cell-free contexts. Subsequent detection of CDDP-peptide interaction by the Tris-Tricine-based electrophoresis revealed that GA-30, a synthetic peptide matching a region of the cytosolic domain of p22phox, could interact with CDDP. These results were further confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis, from which MA-11, an 11-amino acid subdomain of the GA-30 domain, could largely account for the interaction. Amino acid substitutions at Cys50, Met65 and Met73, but not His72, significantly impaired the binding between CDDP and the GA-30 domain, thereby suggesting the potential CDDP-binding residues in p22phox protein. Consistently, the p22phox point mutations at Cys50, Met65 and Met73, but not His72, resensitized OSCC cells to CDDP-induced cytotoxicity and apoptosis. Finally, p22phox might have binding specificity for the platinum drugs, including CDDP, carboplatin and oxaliplatin. Together, we have not only identified p22phox as a novel CDDP-binding protein, but further highlighted the importance of such a drug-protein interaction in drug resistance.

Activation and functional modulation of satellite glial cells by oxaliplatin lead to hyperexcitability of sensory neurons in vitro.

Platinum-based chemotherapeutics still play an important role in cancer therapy, however, severe side effects, such as painful neuropathy, occur frequently. The pathophysiologic mechanisms depend on the applied chemotherapeutic agent and are still controversial. In addition to neuronal damage, disturbance of glial cell activity may contribute to neurotoxicity. Here, we focused on the effect of oxaliplatin on satellite glial cell (SGC) function and on the activity of the dorsal root ganglion (DRG) neurons. SGCs were isolated as high-purity cultures and treated with 1 and 10 µM oxaliplatin for 2, 4 and 24 h. Subsequently, glial fibrillary acid protein (GFAP), reactive oxygen species (ROS), Connexin-43 (Cx-43), and inward rectifier potassium channel 4.1 (Kir4.1) expression was determined by immunocytochemical staining (ICC) and Western blot analyses. Immunochemical staining and Western blot analysis showed an increase in the immune reactivity (IR) and protein levels of ROS, GFAP, and Cx-43. Furthermore, reduction of the IR and protein levels and current density were demonstrated using patch-clamp measurements, of Kir4.1 channels after oxaliplatin exposure. Cytokine release in SGCs was measured using enzyme-linked immunosorbent assays (ELISA) after oxaliplatin exposure and indicated an increased release of IL-6 and TNFα, while IL-1ß was decreased. The direct influence of SGC-secreted factors in the supernatant after oxaliplatin treatment led to the hyperexcitability of cultured DRG neurons. In summary, oxaliplatin has a direct impact on the modulation and function of different SGC proteins. Furthermore, SGC-released factors influence the excitability of sensory neurons, qualifying SGCs as potential targets for the prevention and treatment of oxaliplatin-induced polyneuropathy.