Glutathione S-transferase omega class 1 (GSTO1)-associated large extracellular vesicles are involved in tumor-associated macrophage-mediated cisplatin resistance in bladder cancer.

Bladder cancer poses a significant challenge to chemotherapy due to its resistance to cisplatin, especially at advanced stages. Understanding the mechanisms behind cisplatin resistance is crucial for improving cancer therapy. The enzyme glutathione S-transferase omega class 1 (GSTO1) is known to be involved in cisplatin resistance in colon cancer. This study focused on its role in cisplatin resistance in bladder cancer. Our analysis of protein expression in bladder cancer cells stimulated by secretions from tumor-associated macrophages (TAMs) showed a significant increase in GSTO1. This prompted further investigation into the role of GSTO1 in bladder cancer. We found a strong correlation between GSTO1 expression and cisplatin resistance. Mechanistically, GSTO1 triggered the release of large extracellular vesicles (EVs) that promoted cisplatin efflux, thereby reducing cisplatin-DNA adduct formation and enhancing cisplatin resistance. Inhibition of EV release effectively counteracted the cisplatin resistance associated with GSTO1. In conclusion, GSTO1-mediated EV release may contribute to cisplatin resistance caused by TAMs in bladder cancer. Strategies to target GSTO1 could potentially improve the efficacy of cisplatin in treating bladder cancer.

Phytogalactolipids activate humoral immunity against colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most lethal cancer in the world, and its incidence is steadily rising. In this study, we investigated the induction of humoral immunity by a phytogalactolipid enriched fraction (CRA) derived from the medicinal plant Crassocephalum rabens (Benth.) S. Moore to combat CRC. METHODS: Immunocompetent BALB/c mice were used to evaluate CRA's therapeutic effects in CRC. The phenotypes of B cell subsets in splenocytes and tumors from the CRA-treated mice were isolated and analyzed by flow cytometry. The titers, isotypes, specificity, antigen recognition, and cytotoxic activity of CRA-induced anti-tumor antibodies were determined. The mechanisms of CRA on B cell differentiation were determined by cell-based analyses, including co-cultural with T cells, cytokine analysis, gene expression by qPCR, and protein expression by western blotting. RESULTS: CRA efficiently inhibited tumor growth in colorectal tumor-bearing allograft mice. CRA treatment attracted an abundance of B cells into the tumor consequently enhancing the anti-tumor antibodies in sera and inducing a class-switch. CRA-induced antisera (designated CRA antisera) specifically recognized surface antigens on the plasma membrane of cancer cells. CRA antisera induced cytotoxicity including antibody-dependent cell cytotoxicity, phagocytosis, and complement-dependent cytotoxicity. CRA interacted with IL-6 receptor to activate STAT3 and cMaf, resulting in T cell secretion of IL-21, which, in turn induced B cell differentiation through the IL-21R/STAT3/Blimp-1 pathway. CONCLUSIONS: CRA regulated T cell activity resulting in B cell activation and triggering of anti-tumor antibodies to impede CRC progression.

Changes in the Proteome Profile of A549 Cells Following Helichrysetin-Induced Apoptosis Suggest the Involvement of DNA Damage Response and Cell Cycle Arrest-Associated Proteins.

Our previous findings demonstrated that Helichrysetin possessed promising anti-cancer activity. It was able to induce apoptosis in the A549 cell line. However, its mechanism of action is unknown. The present study aimed to unravel possible underlying molecular mechanisms of helichrysetin-induced apoptosis in A549 (human lung carcinoma) cells using comparative quantitative proteomics (iTRAQ labeled), followed by an exhaustive bioinformatics analysis. Our results suggested that DNA damage response (DDR) and cell cycle arrest were responsible for lung cancer cell death with helichrysetin treatment. Among proteins that changed in abundance were Nrf2 and HMOX1. They are oxidative stress-related proteins and were increased in abundance. BRAT1 was also increased in abundance, suggesting an increase in DNA damage repair, indicating the occurrence of DNA damage due to oxidative stress. However, several essential DDR downstream proteins such as p-ATM, BRCA1, FANCD2, and Rb1 that would further increase DNA damage were found to be dramatically decreased in relative abundance. Cell cycle-related proteins, p53, p21, and cyclin D1, were increased while cyclin A, cyclin E, and cdk2 were decreased. This is predicted to facilitate S-phase arrest. Furthermore, excessive DNA damage and prolonged arrest would in turn result in the induction of mitochondrial-mediated apoptosis. Based on these observations, we postulate that the effects of helichrysetin were in part via the suppression of DNA damage response which led to DNA damage and prolonged cell cycle arrest. Subsequently, this event initiated mitochondrial-mediated apoptosis in A549 lung cancer cells.

MicroRNA-Mediated Mitochondrial Dysfunction Is Involved in the Anti-triple-Negative Breast Cancer Cell Activity of Phytosesquiterpene Lactones.

Aims: Emerging evidence suggests that modulating redox homeostasis through targeting mitochondrial functions may be a useful strategy for suppressing triple-negative breast cancer (TNBC) activities. However, whether there are specific microRNAs (miRNAs) involved in regulating oxidative stress-associated mitochondrial functions that can act as therapeutic targets to suppress TNBC activities remains unclear. Here, we aimed to identify the role of redox-associated miRNAs in TNBC and investigated their potential as therapeutic targets. Results: We identified oxidative stress-responsive differentially expressed miRNAs (DEMs) regulated by phytosesquiterpene lactone deoxyelephantopin (DET) and its novel derivative DETD-35, which are known to inhibit TNBC growth and metastasis in vitro and in vivo, using comparative miRNA microarray analysis and reactive oxygen species (ROS) scavenging approaches. Mitochondrial dysfunction was identified as a major biological function regulated by a few specific DEMs. In particular, miR-4284 was identified to play a role in DET- and DETD-35-mediated ROS production, mitochondrial basal proton leak, and antiproliferation activity in TNBC cells. Moreover, DET- and DETD-35-induced mitochondrial DNA damage was observed in TNBC cells and xenograft tumors. miR-4284 was also identified to play a role in oxidative DNA damage in TNBC tumors. Innovation: We identified a novel role for miR-4284 in regulating mitochondrial basal proton leak in TNBC cells, and highlighted its significance in TNBC tumor oxidative DNA damage, and its direct correlation with TNBC patient survival. Conclusion: We used DET and DETD-35 as proof of concept to demonstrate that activities of anticancer agents can involve regulation of multiple miRNAs playing different roles in cancer progression. Antioxid. Redox Signal. 38, 198-214.

Cellular and Molecular Signaling as Targets for Cancer Vaccine Therapeutics.

Plenty of evidence has recently shown that various inflammatory activities at the local tissue, organ, or even the whole body (systemic) level are strongly linked to many life-threatening chronic diseases, most notably various cancers. However, only very limited information is available for making good use of our supporting immune-modulatory therapeutics for the treatment of cancers. This may result from a lack of studies on specific remedies for efficacious control or modulatory suppression of inflammation-related cancerous diseases. Our group and laboratories were fortunate to have initiated and consistently pursued an integrated team-work program project, aimed at investigating selected medicinal herbs and the derived, purified phytochemical compounds. We focused on the study of key and specific immune-signaling mechanisms at the cellular and molecular levels. We were fortunate to obtain a series of fruitful research results. We believe that our key findings reported herein may be helpful for proposing future thematic and integrated research projects that aim to develop future phytochemical drugs against cancers. The mechanisms of the cellular and molecular systems involved in inflammation are becoming increasingly recognized as keystones for the development of future therapeutic approaches for many chronic and cancerous diseases. Recently, the immune checkpoint inhibitors such as antibodies against PD-1 and/or PD-L1 have been shown to be too expensive for general clinical use, and their effects far from optimal, often showing little or no effect or only short-term efficacy. These results point to the need for developing future immune-regulatory or modulatory therapeutics.

Phyto-sesquiterpene lactones DET and DETD-35 induce ferroptosis in vemurafenib sensitive and resistant melanoma via GPX4 inhibition and metabolic reprogramming.

Acquired resistance to vemurafenib (PLX4032) is a thorny issue in BRAFV600E mutant melanoma therapy. Ferroptotic programmed cell death is a potential strategy for combating therapy-resistant cancers. This study uncovers the adaptation and abnormal upregulation of PUFAs and bioactive oxylipin metabolism in PLX4032 resistant melanoma cells. Phyto-sesquiterpene lactone, DET, and its derivative, DETD-35, induced lipid ROS accumulation and triggered ferroptotic cell death in PLX4032 sensitive (A375) and resistant (A375-R) BRAFV600E melanoma cells by reprogramming glutathione and primary metabolisms, lipid/oxylipin metabolism, and causing mitochondrial damage in which DETD-35 showed superior efficiency to DET. We discovered that DET and DETD-35 are a new type of GPX4 enzyme inhibitor through non-covalent binding. This study provides new insight into the therapeutic mechanisms of both DET and DETD-35 to combat PLX4032 sensitive/resistant BRAFV600E mutant melanomas via targeting GPX4 and ferroptosis.

Current Advancements of Plant-Derived Agents for Triple-Negative Breast Cancer Therapy through Deregulating Cancer Cell Functions and Reprogramming Tumor Microenvironment.

Triple-negative breast cancer (TNBC) is defined based on the absence of estrogen, progesterone, and human epidermal growth factor receptor 2 receptors. Currently, chemotherapy is the major therapeutic approach for TNBC patients; however, poor prognosis after a standard chemotherapy regimen is still commonplace due to drug resistance. Abnormal tumor metabolism and infiltrated immune or stromal cells in the tumor microenvironment (TME) may orchestrate mammary tumor growth and metastasis or give rise to new subsets of cancer cells resistant to drug treatment. The immunosuppressive mechanisms established in the TME make cancer cell clones invulnerable to immune recognition and killing, and turn immune cells into tumor-supporting cells, hence allowing cancer growth and dissemination. Phytochemicals with the potential to change the tumor metabolism or reprogram the TME may provide opportunities to suppress cancer metastasis and/or overcome chemoresistance. Furthermore, phytochemical intervention that reprograms the TME away from favoring immunoevasion and instead towards immunosurveillance may prevent TNBC metastasis and help improve the efficacy of combination therapies as phyto-adjuvants to combat drug-resistant TNBC. In this review, we summarize current findings on selected bioactive plant-derived natural products in preclinical mouse models and/or clinical trials with focus on their immunomodulatory mechanisms in the TME and their roles in regulating tumor metabolism for TNBC prevention or therapy.

Phytogalactolipid dLGG Inhibits Mouse Melanoma Brain Metastasis through Regulating Oxylipin Activity and Re-Programming Macrophage Polarity in the Tumor Microenvironment.

Current conventional cancer therapies for melanoma brain metastasis (MBM) remain ineffective. In this study, we demonstrated the bioefficacy of a phyto-glyceroglycolipid, 1,2-di-O-α-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG) alone, or in combination with liposomal doxorubicin (Lip-DOX) or Avastin against MBM in a syngeneic B16BM4COX-2/Luc brain-seeking melanoma mouse model. Treatment with dLGG-10, dLGG-25, dLGG-10 + Avastin-5, Lipo-DOX-2, dLGG-10 + Lipo-DOX-2 or Lipo-DOX-2 + Avastin-5 suppressed, respectively, 17.9%, 59.1%, 55.7%, 16.2%, 44.5% and 72.4% of MBM in mice relative to the untreated tumor control. Metastatic PD-L1+ melanoma cells, infiltration of M2-like macrophages and CD31+ endothelial cells, and high expression levels of 15-LOX/CYP450 4A enzymes in the brain tumor microenvironment of the tumor control mice were significantly attenuated in dLGG-treated mice; conversely, M1-like resident microglia and cytotoxic T cells were increased. A lipidomics study showed that dLGG promoted B16BM4 cells to secrete oxylipins 9,10-/12,13-EpOMEs into the culture medium. Furthermore, the conditioned medium of B16BM4 cells pretreated with dLGG or 9,10-EpOMEs + 12,13-EpOMEs drove M2-like macrophages to polarize into M1-like macrophages in vitro. An ex vivo 3D-culture assay further demonstrated that dLGG, 9,10-EpOME or 9,10-EpOME + 12,13-EpOME pretreatment attenuated B16BM4 cells invading brain tissue, and prevented microglia/macrophages infiltrating into the interface of melanoma plug and brain organ/tissue. In summary, this report provides a novel therapeutic strategy and mechanistic insights into phytogalactolipid dLGG for combating MBM.

Sesquiterpene Lactone Deoxyelephantopin Isolated from Elephantopus scaber and Its Derivative DETD-35 Suppress BRAFV600E Mutant Melanoma Lung Metastasis in Mice.

Melanoma is a highly metastatic disease with an increasing rate of incidence worldwide. It is treatment refractory and has poor clinical prognosis; therefore, the development of new therapeutic agents for metastatic melanoma are urgently required. In this study, we created a lung-seeking A375LM5IF4g/Luc BRAFV600E mutant melanoma cell clone and investigated the bioefficacy of a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel semi-synthetic derivative, DETD-35, in suppressing metastatic A375LM5IF4g/Luc melanoma growth in vitro and in a xenograft mouse model. DET and DETD-35 treatment inhibited A375LM5IF4g/Luc cell proliferation, and induced G2/M cell-cycle arrest and apoptosis. Furthermore, A375LM5IF4g/Luc exhibited clonogenic, metastatic and invasive abilities, and several A375LM5IF4g/Luc metastasis markers, N-cadherin, MMP2, vimentin and integrin α4 were significantly suppressed by treatment with either compound. Interestingly, DET- and DETD-35-induced Reactive Oxygen Species (ROS) generation and glutathione (GSH) depletion were found to be upstream events important for the in vitro activities, because exogenous GSH supplementation blunted DET and DETD-35 effects on A375LM5IF4g/Luc cells. DET and DETD-35 also induced mitochondrial DNA mutation, superoxide production, mitochondrial bioenergetics dysfunction, and mitochondrial protein deregulation. Most importantly, DET and DETD-35 inhibited lung metastasis of A375LM5IF4g/Luc in NOD/SCID mice through inhibiting pulmonary vascular permeability and melanoma cell (Mel-A+) proliferation, angiogenesis (VEGF+, CD31+) and EMT (N-cadherin) in the tumor microenvironment in the lungs. These findings indicate that DET and DETD-35 may be useful in the intervention of lung metastatic BRAFV600E mutant melanoma.

Association of Arachidonic Acid-derived Lipid Mediators with Subsequent Onset of Acute Myocardial Infarction in Patients with Coronary Artery Disease.

Polyunsaturated fatty acids (PUFAs) have been suggested for cardiovascular health. This study was conducted to investigate the prognostic impacts of the PUFA metabolites, oxylipins, on clinical outcomes in coronary artery disease (CAD). A total of 2,239 patients with stable CAD were prospectively enrolled and followed up regularly. Among them, twenty-five consecutive patients with new onset of acute myocardial infarction (AMI) within 2-year follow-up were studied. Another 50 gender- and age-matched patients without clinical cardiovascular events for more than 2 years were studied for control. Baseline levels of specific arachidonic acid metabolites were significantly higher in patients with subsequent AMI than in the controls. In Kaplan-Meier analysis, the incidence of future AMI was more frequently seen in patients with higher baseline levels of 8-hydroxyeicosatetraenoic acid (HETE), 9-HETE, 11-HETE, 12-HETE, 15-HETE, 19-HETE, 20-HETE, 5,6-epoxyeicosatrienoic acid (EET), 8,9-EET, 11,12-EET, or 14-15-EET when compared to their counterparts (all the P < 0.01). Further, serum levels of these specific HETEs, except for 11,12-EET, were positively correlated to the levels of some inflammatory and cardiac biomarker such as tumor necrosis factor-α and N-terminal pro B-type natriuretic peptide. Accordingly, serum specific oxylipins levels are increased and associated with the consequent onset of AMI, suggesting their potential role for secondary prevention in clinically stable CAD.

White sweet potato ameliorates hyperglycemia and regenerates pancreatic islets in diabetic mice.

BACKGROUND: White sweet potato (WSP) has many potential beneficial effects on metabolic control and on diabetes-related insulin resistance. The antihyperglycemic effects of Tainung No. 10 (TNG10), a variety of WSP in Taiwan, warrant investigation. OBJECTIVE: To investigate the antidiabetic activity of WSP (Ipomoea batatas L. TNG10) and the mechanisms for interventions using whole leaves or tubers of WSP in diabetic mice. DESIGN: Mice were co-administered with streptozotocin and nicotinamide to induce diabetes and then treated with an experimental diet including either 10% WSP tuber (10%-T) and 30% WSP tuber (30%-T) or 0.5% WSP leaf (0.5%-L) and 5% WSP leaf (5%-L). After 8 weeks' treatment, their plasma glycemic parameters, lipid profiles, and inflammatory marker were analyzed. Their pancreases were removed for histopathologic image analysis; proteins were also extracted from their muscles for phosphoinositide 3-kinase pathway analysis. RESULTS: The 30%-T or 5%-L mice had lower plasma glucose, insulin, glucose area under the curve (AUC), homeostatic model assessment of insulin resistance (HOMA-IR), alanine transaminase, triglyceride, and tumor necrosis factor alpha levels. In all diabetic mice, their Langerhans's area was reduced by 60%; however, after 30% WSP-T or 5% WSP-L diets, the mice demonstrated significant restoration of the Langerhans's areas (approximately 30%). Only in 5%-L mice, slightly increased expression of insulin-signaling pathway-related proteins, phosphorylated insulin receptor and protein kinase B and membrane glucose transporter 4 was noted. CONCLUSIONS: WSP has antihyperglycemic effects by inducing pancreatic islet regeneration and insulin resistance amelioration. Therefore, WSP has potential applications in dietary diabetes management.

Deregulating the CYP2C19/Epoxy-Eicosatrienoic Acid-Associated FABP4/FABP5 Signaling Network as a Therapeutic Approach for Metastatic Triple-Negative Breast Cancer.

Recurrence and metastasis are the main causes of triple-negative breast cancer (TNBC) mortality. On the basis of our clinical cohorts and integrative omics analyses, we hypothesized that understanding the interplay between fatty acid binding protein (FABP) and epoxy-eicosatrienoic acid (EET) driven metastatic progression can uncover a new opportunity for TNBC intervention. In this study, the biological relevance of increased protein expression of CYP2C19, FABP4, and FABP5 in TNBC tumors and in the TNBC cell line (MDA-MB-231), as well as its highly metastatic lung seeking variant (LM6) were delineated from publicly available datasets, shRNA-mediated knockdown, EET supplementation, cancer and stromal cell co-cultures, and an orthotopic and resection xenograft tumor mouse model. We found that the high expression levels of CYP2C19 and FABP4 and FABP5 are critical in TNBC metastatic transformation and stromal cell interactions. Furthermore, EET-associated nuclear translocation of FABP4 and FABP5 and nuclear accumulation of SREBP-2 or PPAR-γ influence TNBC cell proliferation, migratory transformation, and distal metastasis priming. Most notably, we uncovered novel bioefficacy and modes of action of the anticancer drug doxorubicin and a phytogalactolipid, 1,2-di-O-α-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG), which effectively attenuated TNBC recurrence and lung metastasis through deregulating the FABP/EET dynamics and levels. This study, therefore, introduces a novel approach to combating TNBC by targeting the FABP/EET/CYP-associated metastatic signaling network.

Essential Oil of Mentha aquatica var. Kenting Water Mint Suppresses Two-Stage Skin Carcinogenesis Accelerated by BRAF Inhibitor Vemurafenib.

The v-raf murine sarcoma viral homolog B1 (BRAF) inhibitor drug vemurafenib (PLX4032) is used to treat melanoma; however, epidemiological evidence reveals that it could cause cutaneous keratoacanthomas and squamous cell carcinoma in cancer patients with the most prevalent HRASQ61L mutation. In a two-stage skin carcinogenesis mouse model, the skin papillomas induced by 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) (DT) resemble the lesions in BRAF inhibitor-treated patients. In this study, we investigated the bioactivity of Mentha aquatica var. Kenting Water Mint essential oil (KWM-EO) against PDV cells, mouse keratinocytes bearing HRASQ61L mutation, and its effect on inhibiting papilloma formation in a two-stage skin carcinogenesis mouse model with or without PLX4032 co-treatment. Our results revealed that KWM-EO effectively attenuated cell viability, colony formation, and the invasive and migratory abilities of PDV cells. Induction of G2/M cell-cycle arrest and apoptosis in PDV cells was also observed. KWM-EO treatment significantly decreased the formation of cutaneous papilloma further induced by PLX4032 in DT mice (DTP). Immunohistochemistry analyses showed overexpression of keratin14 and COX-2 in DT and DTP skin were profoundly suppressed by KWM-EO treatment. This study demonstrates that KWM-EO has chemopreventive effects against PLX4032-induced cutaneous side-effects in a DMBA/TPA-induced two-stage carcinogenesis model and will be worth further exploration for possible application in melanoma patients.

Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer.

BACKGROUND: Current prognostic tools and targeted therapeutic approaches have limited value for metastatic triple negative breast cancer (TNBC). Building upon current knowledge, we hypothesized that epoxyeicosatrienoic acids (EETs) and related CYP450 epoxygenases may have differential roles in breast cancer signaling, and better understanding of which may uncover potential directions for molecular stratification and personalized therapy for TNBC patients. METHODS: We analyzed the oxylipin metabolome of paired tumors and adjacent normal mammary tissues from patients with pathologically confirmed breast cancer (N = 62). We used multivariate statistical analysis to identify important metabolite contributors and to determine the predictive power of tumor tissue metabolite clustering. In vitro functional assays using a panel of breast cancer cell lines were carried out to further confirm the crucial roles of endogenous and exogenous EETs in the metastasis transformation of TNBC cells. Deregulation of associated downstream signaling networks associated with EETs/CYPs was established using transcriptomics datasets from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Comparative TNBC proteomics using the same tissue specimens subjected to oxylipin metabolomics analysis was used as validation set. RESULTS: Metabolite-by-metabolite comparison, tumor immunoreactivity, and gene expression analyses showed that CYP epoxygenases and arachidonic acid-epoxygenation products, EET metabolites, are strongly associated with TNBC metastasis. Notably, all the 4 EET isomers (5,6-, 8,9-, 11,12-, and 14,15-EET) was observed to profoundly drive the metastasis transformation of mesenchymal-like TNBC cells among the TNBC (basal- and mesenchymal-like), HER2-overexpressing and luminal breast cancer cell lines examined. Our pathway analysis revealed that, in hormone-positive breast cancer subtype, CYP epoxygenase overexpression is more related to immune cell-associated signaling, while EET-mediated Myc, Ras, MAPK, EGFR, HIF-1α, and NOD1/2 signaling are the molecular vulnerabilities of metastatic CYP epoxygenase-overexpressing TNBC tumors. CONCLUSIONS: This study suggests that categorizing breast tumors according to their EET metabolite ratio classifiers and CYP epoxygenase profiles may be useful for prognostic and therapeutic assessment. Modulation of CYP epoxygenase and EET-mediated signaling networks may offer an effective approach for personalized treatment of breast cancer, and may be an effective intervention option for metastatic TNBC patients.

Cumingianoside A, a Phyto-Triterpenoid Saponin Inhibits Acquired BRAF Inhibitor Resistant Melanoma Growth via Programmed Cell Death.

Mutated proto-oncogene BRAF is a bona fide therapeutic target for melanomas. Regrettably, melanoma acquires resistance to BRAF inhibitors, e.g., vemurafenib (PLX4032) casting doubt on this promising melanoma targeted therapy. In this study, we explored the bioactivity of triterpenoid saponin cumingianoside A (CUMA), isolated from leaves and twigs of Dysoxylum cumingianum against PLX4032-resistant BRAFV 600E mutant melanoma A375-R in vitro and in vivo. Our data show that CUMA treatment inhibited A375-R melanoma cell proliferation in a time- and dose-dependent manner. CUMA also suppressed the activity of CDK1/cyclin B1 complex and led to G2/M-phase arrest of A375-R cells. Furthermore, CUMA treatment resulted in induction of apoptosis as shown by the increased activation of caspase 3 and caspase 7, and the proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). We also observed that CUMA induced autophagy-like activity in A375-R cells, as shown by the increased expression of autophagy-related genes and increased formation of autophagosomes. Moreover, we found that CUMA treatment induced ER stress response and co-treatment with an ER stress inhibitor (4-PBA) could attenuate apoptosis induced by CUMA. Importantly, orally administered CUMA as a single agent or in combination with PLX4032 exhibited strong tumor growth inhibition in a PLX4032-resistant A375-R xenograft mouse model, and with little toxicity. This is the first report to explore the anti-tumor activity of CUMA in vitro and in vivo mechanistically, and our results imply that this triterpenoid saponin may be suitable for development into an anti-melanoma agent.

Phyto-sesquiterpene lactone deoxyelephantopin and cisplatin synergistically suppress lung metastasis of B16 melanoma in mice with reduced nephrotoxicity.

BACKGROUND: Cisplatin (CP) is a chemotherapeutic drug for treating melanoma that also causes adverse side effects in cancer patients. PURPOSE: This study investigated the bioefficacy of a phytoagent deoxyelephantopin (DET) in inhibiting B16 melanoma cell activity, its synergism with CP against metastatic melanoma, and its capability to attenuate CP side effects in animals. METHODS: DET and CP bioactivities were assessed by MTT assay, isobologram analysis, time-lapse microscopy, migration and invasion assays, flow cytometry and western blotting. In vivo bioluminescence imaging was used to detect lung metastasis of B16 cells carrying COX-2 reporter gene in syngeneic mice. H&E staining and immunohistochemistry were used to evaluate the compound/drug efficacy and CP side effects. Nephrotoxicity caused by CP treatment in mice was evaluated by UPLC/ESI-QTOF MS - based metabolomics and haematometry. RESULT: DET, alone or in combination with cisplatin, inhibited B16 cell proliferation, migration, and invasion, and induced cell-cycle arrested at the G2/M phase and de-regulated cell-cycle mediators in cancer cells. In a murine B16COX-Luc metastatic allograft model, CP2 (2  mg/kg) treatment inhibited B16 lung metastasis accompanied by severe body weight loss, renal damage and inflammation, and haematological toxicity. DET10 and CP cotreatment (DET10 + CP1) or sequential treatment (CP2→DET10) significantly inhibited formation of pulmonary melanoma foci and reduced renal damage. DET pretreatment (Pre-DET10) or CP2→DET10 treatment had the longest survival (52  vs. 37 days for tumor control mice). CP treatment caused abnormally accumulated urea cycle metabolites and serotonin metabolite hippuric acid in renal tissues that were not seen with DET alone or in combination with CP. CONCLUSION: The CP and DET combination may be an effective intervention for melanoma with reduced side effects.

Plant galactolipid dLGG suppresses lung metastasis of melanoma through deregulating TNF-α-mediated pulmonary vascular permeability and circulating oxylipin dynamics in mice.

This study demonstrates the bioefficacy and gives mechanistic insights into a plant galactolipid 1,2-di-O-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (dLGG) against metastatic melanoma using a syngeneic mouse model implanted with B16COX-2/Luc melanoma. dLGG-20 (p.o. dLGG 20 mg/kg) and anti-cancer drug CP-2 (i.p. cisplatin 2 mg/kg) treatment significantly inhibited lung metastasis of melanoma in mice 91 and 57%, respectively, as determined by bioluminescence intensity. Moreover, dLGG-20 and CP-2 treatment prolonged mouse mean survival time. dLGG-20 treatment significantly inhibited the expression levels of several molecular markers, that is, PCNA, MMP2, COX-2, VEGF, vimentin, snail, TGF-ß, ß-catenin, TNF-α, PD-1 and PD-L1 in mouse lung tissues compared to tumor control mice. Significant inhibition of macrophage and neutrophil infiltration and promotion of CD8 + Tc cell recruitment in the lung microenvironment was observed in dLGG-20-treated mice. A LC/MS-based comparative oxylipin metabolomics study showed that dLGG-20 treatment significantly induced (5.0- to 12.8-fold) the 12/15-LOX catalyzed oxylipin products in mouse serum including 17-HDHA from DHA, 15-HEPE from EPA, 8- and 12-HETEs from AA, and CYP450-derived 20-HETE from AA. CP-2 treatment increased 12/15-LOX derived 8-, 11- and 12-HETEs from AA, and CYP450 derived 11,12-EET from AA ad 9,10-DHOME from LA by 5.3- to 8.1-fold. Of note, dLGG and 17-HDHA were more effective than CP in preventing B16 melanoma cell-induced pulmonary vascular permeability in mice through inhibition of TNF-α production, up-regulation of tight junction proteins claudin1 and ZO-2 and deregulation of Src activation. In conclusion, this study shows the novel therapeutic effect of phytoagent dLGG and suggests its potential as a therapeutic agent for metastatic melanoma.

Dual specificity phosphatase DUSP6 promotes endothelial inflammation through inducible expression of ICAM-1.

Tumor necrosis factor (TNF)-α activates a diverse array of signaling pathways in vascular endothelial cells (ECs), leading to the inflammatory phenotype that contributes to the vascular dysfunction and neutrophil emigration in patients with sepsis. To date, it is not well understood what key regulator might coordinate signaling pathways to achieve inflammatory response in TNF-α-stimulated ECs. This study investigated the role of dual specificity phosphatase-6 (DUSP6) in the regulation of endothelial inflammation. Using knockout mice, we found that DUSP6 is important for TNF-α-induced endothelial intercellular adhesion molecule-1 (ICAM-1) expression in aorta and in vein. Moreover, genetic deletion of Dusp6 in pulmonary circulation significantly alleviated the susceptibility of mice to lung injury caused by neutrophil recruitment during experimental sepsis induced by TNF-α or lipopolysaccharide (LPS). The role of DUSP6 was further investigated in primary human umbilical vein endothelial cells (HUVECs). Employing RNAi approach in which endogenous DUSP6 was ablated, we showed a critical function of DUSP6 to facilitate TNF-α-induced ICAM-1 expression and endothelial leukocyte interaction. Interestingly, DUSP6-promoted endothelial inflammation is independent of extracellular signaling-regulated kinase (ERK) signaling. On the other hand, inducible DUSP6 leads to activation of canonical nuclear factor (NF)-κB-mediated transcription of ICAM-1 gene in TNF-α-stimulated human ECs. These results are the first to demonstrate a positive role of DUSP6 in endothelial inflammation-mediated pathological process and the underlying mechanism through which DUSP6 promotes NF-κB signaling in the inflamed ECs. Our findings suggest that manipulation of DUSP6 holds great potential for the treatment of acute inflammatory diseases.

Phytoagent deoxyelephantopin derivative inhibits triple negative breast cancer cell activity by inducing oxidative stress-mediated paraptosis-like cell death.

Triple negative breast cancer (TNBC) is a highly metastatic cancer among the breast cancer subgroups. A thorny issue for clinical therapy of TNBC is lack of an efficient targeted therapeutic strategy. We previously created a novel sesquiterpene lactone analog (named DETD-35) derived from plant deoxyelephantopin (DET) which exhibits potent effects against human TNBC MDA-MB-231 tumor growth in a xenograft mouse model. Here we studied the mechanisms of both DET and DETD-35 against MDA-MB-231 cells. DETD-35 (3-fold decreased in IC50) exhibited better anti-TNBC cell activity than DET as observed through induction of reactive oxygen species production (within 2 h treatment) and damage to the ER structures, resulting in ER-derived cytoplasmic vacuolation and ubiquitinated protein accumulation in the treated cells. Intriguingly, the effects of both compounds were blockaded by pretreatment with ROS scavengers, N-acetylcysteine and reduced glutathione, and protein synthesis inhibitor, cycloheximide. Further, knockdown of MEK upstream regulator RAF1 and autophagosomal marker LC3, and co-treatment with JNK or ERK1/2 inhibitor resulted in the most significant attenuation of DETD-35-induced morphological and molecular or biochemical changes in cancer cells, while the inhibitory effect of DET was not influenced by MAPK inhibitor treatment. Therefore, DETD-35 exerted both ER stress-mediated paraptosis and apoptosis, which may explain its superior activity to DET against TNBC cells. Although the chemotherapeutic drug paclitaxel induced vacuole-like structures in MDA-MB-231 cells, no paraptotic cell death features were detected. This study provides a strategy for combating TNBC through sesquiterpene lactone analogs by induction of oxidative and ER stresses that cause paraptosis-like cell death.

Phytoagent Deoxyelephantopin and Its Derivative Inhibit Triple Negative Breast Cancer Cell Activity through ROS-Mediated Exosomal Activity and Protein Functions.

A novel plant sesquiterpene lactone derivative, DET derivative (DETD)-35, originating from parental deoxyelephantopin (DET) was previously observed to effectively suppress human triple negative breast cancer (TNBC) MDA-MB-231 cell activity and tumor growth in mice. In this study, the mechanisms underlying the activity of DETD-35 were elucidated. DET and DETD-35 induced reactive oxygen species (ROS) which caused structural damage and dysfunction of mitochondria and increased cytosolic calcium level, subsequently evoking exosome release from the cancer cells. Intriguingly, exosomes induced by both compounds had an atypical function. Cancer cell-derived exosomes commonly show metastatic potential, but upon DET/DETD-35 treatment exosomes showed anti-proliferative activity against MDA-MB-231 cells. Quantitative proteome analysis of TNBC cell-secreted exosomes showed that DET and DETD-35 attenuated the expression of proteins related to cell migration, cell adhesion, and angiogenesis. Furthermore, several exosomal proteins participating in biological mechanisms such as oxidative stress and decrease of transmembrane potential of mitochondria were found deregulated by treatment with either compound. Pretreatment with ROS scavenger, N-acetylcysteine, blockaded DET- or DETD-35-induced oxidative stress and calcium dependent exosome release mechanisms, and also reverted DET- or DETD-35-induced reprogramming exosomal protein expression profiles resulting in attenuation of exosomal toxicity against TNBC cell proliferation. In summary, this study shows that a plant-derived sesquiterpene lactone DET and its analog DETD-35 inhibitory TNBC cell activities through oxidative stress-induced cancer cell releasing exosomes in tandem with alteration of exosomal protein composition and functions. The findings of this study suggest that DETD-35 may be suitable for further development into an anti-TNBC drug.