Isoorientin reverses lung cancer drug resistance by promoting ferroptosis via the SIRT6/Nrf2/GPX4 signaling pathway.

Cisplatin, or DDP, is a highly successful and well-known chemotherapy drug used to treat cancer. Acquired resistance to chemotherapy is a major clinical concern, yet the mechanisms of this resistance are still unknown. Ferroptosis is a type of cell death distinct from other forms, fueled by a buildup of iron-associated lipid reactive oxygen species (ROS). Gaining insight into the process of ferroptosis could lead to novel treatments for overcoming cancer resistance. In this study, the combination of isoorientin (IO) and DDP treatment resulted in a significant decrease in the viability of drug-resistant cells, a substantial increase in intracellular iron, malondialdehyde (MDA) and ROS concentrations, a notable decrease in glutathione concentration, and the occurrence of ferroptosis in cells, as revealed by in vitro and in vivo experiments. Additionally, there was a decrease in the expression of nuclear factor-erythroid factor 2-related factor 2 (Nrf2), glutathione peroxidase 4 (GPX4), and sirtuin 6 (SIRT6) proteins, and an increase in cellular ferroptosis. Isoorientin acts as a mediator to regulate cellular ferroptosis and reverse drug resistance in lung cancer cells by controlling the SIRT6/Nrf2/GPX4 signaling pathway. The findings of this study suggest that IO can promote ferroptosis and reverse drug resistance in lung cancer through the SIRT6/Nrf2/GPX4 signaling pathway, thus offering a theoretical basis for its potential clinical application.

Effective prediction of potential ferroptosis critical genes in clinical colorectal cancer.

Background: Colon cancer is common worldwide, with high morbidity and poor prognosis. Ferroptosis is a novel form of cell death driven by the accumulation of iron-dependent lipid peroxides, which differs from other programmed cell death mechanisms. Programmed cell death is a cancer hallmark, and ferroptosis is known to participate in various cancers, including colon cancer. Novel ferroptosis markers and targeted colon cancer therapies are urgently needed. To this end, we performed a preliminary exploration of ferroptosis-related genes in colon cancer to enable new treatment strategies. Methods: Ferroptosis-related genes in colon cancer were obtained by data mining and screening for differentially expressed genes (DEGs) using bioinformatics analysis tools. We normalized the data across four independent datasets and a ferroptosis-specific database. Identified genes were validated by immunohistochemical analysis of pathological and healthy clinical samples. Results: We identified DEGs in colon cancer that are involved in ferroptosis. Among these, five core genes were found: ELAVL1, GPX2, EPAS1, SLC7A5, and HMGB1. Bioinformatics analyses revealed that the expression of all five genes, except for EPAS1, was higher in tumor tissues than in healthy tissues. Conclusions: The preliminary exploration of the five core genes revealed that they are differentially expressed in colon cancer, playing an essential role in ferroptosis. This study provides a foundation for subsequent research on ferroptosis in colon cancer.

Down-regulating Nrf2 by tangeretin reverses multiple drug resistance to both chemotherapy and EGFR tyrosine kinase inhibitors in lung cancer.

Multiple drug resistance (MDR) is the major obstacle for both chemotherapy and molecular-targeted therapy for cancer, which is mainly caused by overexpression of ABC transporters or genetic mutation of drug targets. Based on previous studies, we hypothesized that ROS/Nrf2 is the common target for overcoming acquired drug resistance to both targeted therapy and chemotherapy treatments. In this study, we firstly proved that the levels of ROS and Nrf2 were remarkably up-regulated in both H1975 (Gefitinib-resistant lung cancer cells with T790M) and A549/T (paclitaxel-resistant) cells, which is consistent with the clinical database analysis results of lung cancer patients that Nrf2 expression level is negatively related to survival rate. Nrf2 Knockdown with siRNA or tangeretin (TG, a flavonoid isolated from citrus peels) inhibited the MDR cell growth by suppressing the Nrf2 pathway, and efficiently enhanced the anti-tumor effects of paclitaxel and AZD9291 (the third generation of TKI) in A549/T or H1975, respectively. Moreover, TG sensitized A549/T cells-derived xenografts to paclitaxel via inhibiting Nrf2 and its downstream target P-gp, leading to an increased paclitaxel concentration in tumors. Collectively, targeting Nrf2 to enhance ROS may be a common target for overcoming the acquired drug resistance and enhancing the therapeutic effects of chemotherapy and molecular-targeted therapy.

The combination of Lonicerae Japonicae Flos and Forsythiae Fructus herb-pair alleviated inflammation in liver fibrosis.

Objective: To explore the active components and epigenetic regulation mechanism underlying the anti-inflammatory effects of Lonicerae Japonicae Flos and Forsythiae Fructus herb-pair (LFP) in carbon tetrachloride (CCl4)-induced rat liver fibrosis. Methods: The main active ingredients and disease-related gene targets of LFP were determined using TCMSP and UniProt, and liver fibrosis disease targets were screened in the GeneCards database. A network was constructed with Cytoscape 3.8.0 and the STRING database, and potential protein functions were analyzed using bioinformatics analysis. Based on these analyses, we determined the main active ingredients of LFP and evaluated their effects in a CCl4-induced rat liver fibrosis model. Serum biochemical indices were measured using commercial kits, hepatocyte tissue damage and collagen deposition were evaluated by histopathological studies, and myofibroblast activation and inflammation were detected by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. High-performance liquid chromatography-mass spectrometry was performed to determine the levels of homocysteine, reduced glutathione, and oxidized glutathione, which are involved in inflammation and oxidative stress. Results: The main active components of LFP were quercetin, kaempferol, and luteolin, and its main targets were α-smooth muscle actin, cyclooxygenase-2, formyl-peptide receptor-2, prostaglandin-endoperoxide synthase 1, nuclear receptor coactivator-2, interleukinß, tumor necrosis factor α, CXC motif chemokine ligand 14, and transforming growth factor ß1. A combination of quercetin, kaempferol, and luteolin alleviated the symptoms of liver fibrosis. Conclusion: The results of this study support the role of LFP in the treatment of liver fibrosis, and reveal that LFP reduces collagen formation, inflammation, and oxidative stress. This study suggests a potential mechanism of action of LFP in the treatment of liver fibrosis.

Nobiletin Induces Ferroptosis in Human Skin Melanoma Cells Through the GSK3ß-Mediated Keap1/Nrf2/HO-1 Signalling Pathway.

Melanoma is an aggressive malignant skin tumour with an increasing global incidence. However, current treatments have limitations owing to the acquired tumour drug resistance. Ferroptosis is a recently discovered form of programmed cell death characterised by iron accumulation and lipid peroxidation and plays a critical role in tumour growth inhibition. Recently, ferroptosis inducers have been regarded as a promising therapeutic strategy to overcome apoptosis resistance in tumour cells. In this study, we reported that nobiletin, a natural product isolated from citrus peel, and exhibited antitumour activity by inducing ferroptosis in melanoma cells. Subsequently, we further explored the potential mechanism of nobiletin-induced ferroptosis, and found that the expression level of glycogen synthase kinase 3ß (GSK3ß) in the skin tissue of patients with melanoma was significantly reduced compared to that in the skin of normal tissue. Additionally, nobiletin increased GSK3ß expression in melanoma cells. Moreover, the level of Kelch-like Ech-associated protein-1 (Keap1) was increased, while the level of nuclear factor erythroid 2-related factor 2 (Nrf2), and haem oxygenase-1 (HO-1) was decreased in nobiletin-treated melanoma cells, suggesting that the antioxidant defence system was downregulated. Furthermore, knockdown of GSK3ß significantly reduced nobiletin-induced ferroptosis and upregulated the Keap1/Nrf2/HO-1 signalling pathway, while the opposite was observed in cells overexpressing GSK3ß. In addition, molecular docking assay results indicated that nobiletin showed strong binding affinities for GSK3ß, Keap1, Nrf2, and HO-1. Taken together, our results demonstrated that nobiletin could induce ferroptosis by regulating the GSK3ß-mediated Keap1/Nrf2/HO-1 signalling pathway in human melanoma cells. Hence, nobiletin stands as a promising drug candidate for melanoma treatment with development prospects.

A multiple-targets alkaloid nuciferine overcomes paclitaxel-induced drug resistance in vitro and in vivo.

OBJECTIVE: Multidrug resistance (MDR) is the major barrier to the successful treatment of chemotherapy. Compounds from nature products working as MDR sensitizers provided new treatment strategies for chemo-resistant cancers patients. METHODS: We investigated the reversal effects of nuciferine (NF), an alkaloid from Nelumbo nucifera and Nymphaea caerulea, on the paclitaxel (PTX) resistance ABCB1-overexpressing cancer in vitro and in vivo, and explored the underlying mechanism by evaluating drug sensitivity, cell cycle perturbations, intracellular accumulation, function and protein expression of efflux transporters as well as molecular signaling involved in governing transporters expression and development of MDR in cancer. RESULTS: NF overcomes the resistance of chemotherapeutic agents included PTX, doxorubicin (DOX), docetaxel, and daunorubicin to HCT-8/T and A549/T cancer cells. Notably, NF suppressed the colony formation of MDR cells in vitro and the tumor growth in A549/T xenograft mice in vivo, which demonstrated a very strong synergetic cytotoxic effect between NF and PTX as combination index (CI) (CI<0.1) indicated. Furthermore, NF increased the intracellular accumulation of P-gp substrates included DOX and Rho123 in the MDR cells and inhibited verapamil-stimulated ATPase activity. Mechanistically, inhibition of PI3K/AKT/ERK pathways by NF suppressed the activation of Nrf2 and HIF-1α, and further reduced the expression of P-gp and BCRP, contributing to the sensitizing effects of NF against MDR in cancer. CONCLUSION: This novel finding provides a promising treatment strategy for overcoming MDR and improving the efficiency of chemotherapy by using a multiple-targets MDR sensitizer NF.

Ginsenoside Rg5 overcomes chemotherapeutic multidrug resistance mediated by ABCB1 transporter: in vitro and in vivo study.

BACKGROUND: Multidrug resistance (MDR) to chemotherapy drugs remains a major challenge in clinical cancer treatment. Here we investigated whether and how ginsenoside Rg5 overcomes the MDR mediated by ABCB1 transporter in vitro and in vivo. METHODS: Cytotoxicity and colon formation as well as the intracellular accumulation of ABCB1 substrates were carried out in MDR cancer cells A2780/T and A549/T for evaluating the reversal effects of Rg5. The expressions of ABCB1 and Nrf2/AKT pathway were determined by Western blotting. An A549/T cell xenograft model was established to investigate the MDR reversal activity of Rg5 in vivo. RESULTS: Rg5 significantly reversed ABCB1-mediated MDR by increasing the intracellular accumulation of ABCB1 substrates without altering protein expression of ABCB1. Moreover, Rg5 activated ABCB1 ATPase and reduced verapamil-stimulated ATPase activity, suggesting a high affinity of Rg5 to ABCB1 binding site which was further demonstrated by molecular docking analysis. In addition, co-treatment of Rg5 and docetaxel (TXT) suppressed the expression of Nrf2 and phosphorylation of AKT, indicating that sensitizing effect of Rg5 associated with AKT/Nrf2 pathway. In nude mice bearing A549/T tumor, Rg5 and TXT treatment significantly suppressed the growth of drug-resistant tumors without increase in toxicity when compared to TXT given alone at same dose. CONCLUSION: Therefore, combination therapy of Rg5 and chemotherapy drugs is a strategy for the adjuvant chemotherapy, which encourages further pharmacokinetic and clinical studies.

Nobiletin and its derivatives overcome multidrug resistance (MDR) in cancer: total synthesis and discovery of potent MDR reversal agents.

Our recent studies demonstrated that the natural product nobiletin (NOB) served as a promising multidrug resistance (MDR) reversal agent and improved the effectiveness of cancer chemotherapy in vitro. However, low aqueous solubility and difficulty in total synthesis limited its application as a therapeutic agent. To tackle these challenges, NOB was synthesized in a high yield by a concise route of six steps and fourteen derivatives were synthesized with remarkable solubility and efficacy. All the compounds showed improved sensitivity to paclitaxel (PTX) in P-glycoprotein (P-gp) overexpressing MDR cancer cells. Among them, compound 29d exhibited water solubility 280-fold higher than NOB. A drug-resistance A549/T xenograft model showed that 29d, at a dose of 50 mg/kg co-administered with PTX (15 mg/kg), inhibited tumor growth more effective than NOB and remarkably increased PTX concentration in the tumors via P-gp inhibition. Moreover, Western blot experiments revealed that 29d inhibited expression of NRF2, phosphorylated ERK and AKT in MDR cancer cells, thus implying 29d of multiple mechanisms to reverse MDR in lung cancer.

Nobiletin potentiates paclitaxel anticancer efficacy in A549/T xenograft model: Pharmacokinetic and pharmacological study.

BACKGROUND: Nobiletin (N), a polymethoxylated flavone from citrus fruits, enhanced anti-cancer effects of paclitaxel (PTX) in multi-drug resistance (MDR) cancer cells via inhibiting P-glycoprotein (P-gp) in our previous report. But the in vivo chemo-sensitizing effect of nobiletin is unknown. Moreover, considering the nonlinear pharmacokinetics and narrow therapeutic window of PTX, drug-drug interaction should be explored for using nobiletin with PTX together. PURPOSE: In this study, we wanted to explore whether nobiletin could affect the pharmacokinetic (PK) behavior of PTX and reverse drug resistance in vivo as well as the corresponding mechanisms. STUDY DESIGN AND METHODS: Accurate and sensitive UPLC-MS/MS method was developed for the detection of PTX, and was applied to the pharmacokinetic study in rats. In vivo anti-MDR tumor study was carried out with A549/T xenograft nude mice model. Immunohistochemistry and western blot analysis were used for evaluating the levels of P-gp, Nrf2, and AKT/ERK pathways in MDR tumors. RESULTS: Nobiletin significantly enhanced the therapeutic effects of PTX, and inhibited the MDR tumor sizes in the A549/T xenograft model, while PTX or nobiletin alone did not. We found that nobiletin increased the PTX concentrations in tumor tissues but did not affect the PK behavior of PTX. Notably, Nrf2 and phosphorylation of AKT/ERK expression in MDR tumor tissues were significantly inhibited by giving nobiletin and PTX together. However, nobiletin did not affect the expression of P-gp. CONCLUSION: Nobiletin reversed PTX resistance in MDR tumor via increasing the PTX content in the MDR tumor and inhibiting AKT/ERK/Nrf2 pathways, but without affecting the systematic exposure of PTX, indicating that nobiletin may be an effective and safe MDR tumor reversal agent.

Role of tangeretin as a potential bioavailability enhancer for silybin: Pharmacokinetic and pharmacological studies.

Biological responses of a variety of naturally occurring compounds in vivo were restrained by their poor oral bioavailability. Silybin, as one of the active ingredients of silymarin, has presented promising bioactivity for the treatment of chronic liver diseases and cancer. However, its exposure in body was limited. In this study, silybin was demonstrated to be substrates of both BCRP and MRP2 by utilizing monolayer Caco-2 cell model and confirmed in MDCK cells overexpressing specific efflux transporter. Of all compounds screened, tangeretin, a potent inhibitor of efflux transporters of BCRP, MRP2 and P-gp, was able to enhance exposure of silybin by inhibiting functions of the barriers mediating transcellular transport. Moreover, study carried out in sandwich-cultured rat hepatocyte (SCH) model showed that the biliary excretion index (BEI) and in vitro biliary clearance of silybin decreased as levels of tangeretin increased, indicating efflux transporters mediating biliary excretion of silybin might be involved. Pharmacokinetic behaviors of silybin in rats were altered by co-administration of tangeretin, in terms of increased AUC and Cmax of silybin by comparing with that of silybin given alone. In addition, results coming from CCl4-induced acute liver injury rat model revealed that protection effect of silybin against liver damage in the presence of tangeretin was significantly enhanced. All these data were evident that efflux transporters play a critical role in transcellular transport of silybin and account for its low bioavailability. Enhanced bioavailability of silybin with co-administration of tangeretin by significantly inhibiting the efflux transporters further boost its bioactivity which is of particular importance in clinical use.

Protein kinase C inhibitor chelerythrine selectively inhibits proliferation of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targeted therapy currently. Recent studies imply that protein kinase C may play important roles in TNBC development and could be a specific target. In this study, we evaluated the anti-proliferative activity of PKC inhibitor chelerythrine on a panel of breast cancer cell lines. Chelerythrine selectively inhibited the growth of TNBC cell lines compared to non-TNBC cell lines as demonstrated by in vitro cell proliferation assay and colony formation assay, as well as evidenced by in vivo xenograft assay. The selective anti-proliferative effect of chelerythrine was associated with induction of apoptosis in TNBC cell lines. We further demonstrated that PKN2, one of the PKC subtypes, was highly expressed in TNBC cell lines, and knocking down PKN2 in TNBC cells inhibited colony formation and xenograft growth. This indicates that PKN2 is required for the survival of TNBC cells, and could be the target mediates the selective activity of chelerythrine. Finally, combination of chelerythrine and chemotherapy reagent taxol showed synergistic/additive effect on TNBC cell lines. Our results suggest chelerythrine or other PKC inhibitors may be promising regimens for TNBC tumors.

Neo-tanshinlactone selectively inhibits the proliferation of estrogen receptor positive breast cancer cells through transcriptional down-regulation of estrogen receptor alpha.

Breast cancer, the most frequent cancer in women, is the second leading cause of cancer-related death. Estrogens and estrogen receptors are well recognized to play predominant roles in breast cancer development and growth. Neo-tanshinlactone is a natural product isolated from Salvia miltiorrhiza and showed selective growth inhibition of ER+ breast cancer cell lines as demonstrated by cell proliferation assay and colony formation assay. The selective anti-proliferative effect of neo-tanshinlactone was associated with the induction of apoptosis in ER+ breast cancer cells. We also found that neo-tanshinlactone decreased steady state ESR1 mRNA levels in ER+ breast cancer cells, which was further confirmed by analysis of ER protein levels as well as the mRNA levels of target genes of this transcription factor, such as ESR2, BRCA1, CCND1, GREB1, TFF1, SERPINB9 and ABCA3. Furthermore, analysis of heterogeneous nuclear RNA (hnRNA) demonstrated that neo-tanshinlactone inhibited ESR1 mRNA de novo synthesis. The decrease of steady state ESR1 mRNA upon neo-tanshinlactone treatment was not abolished by protein synthesis inhibitor cycloheximide. And inhibition of mRNA synthesis with actinomycin D revealed no significant effect of neo-tanshinlactone on ESR1 mRNA stability. These results indicated that transcriptional down-regulation of ESR1 mRNA could contribute to the selective activity of neo-tanshinlactone on ER+ breast cancer cells. And as expected, the combination of neo-tanshinlactone and antiestrogen reagent tamoxifen showed a synergistic effect on growth of ER+ MCF7 cells. Our results suggest that neo-tanshinlactone is a promising regimen for ER+ breast tumors.

Tangeretin, a citrus pentamethoxyflavone, antagonizes ABCB1-mediated multidrug resistance by inhibiting its transport function.

Multidrug resistance (MDR) and tumor metastasis are the main causes of chemotherapeutic treatment failure and mortality in cancer patients. In this study, at achievable nontoxic plasma concentrations, citrus flavonoid tangeretin has been shown to reverse ABCB1-mediated cancer resistance to a variety of chemotherapeutic agents effectively. Co-treatment of cells with tangeretin and paclitaxel activated apoptosis as well as arrested cell cycle at G2/M-phase. Tangeretin profoundly inhibited the ABCB1 transporter activity since it significantly increased the intracellular accumulation of doxorubicin, and flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the expression of ABCB1. Moreover, it stimulated the ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. The molecular docking results indicated a favorable binding of tangeretin with the transmemberane region site 1 of homology modeled ABCB1 transporter. The overall results demonstrated that tangeretin could sensitize ABCB1-overexpressing cancer cells to chemotherapeutical agents by directly inhibiting ABCB1 transporter function, which encouraged further animal and clinical studies in the treatment of resistant cancers.

In Vitro Anticancer Activity of a Nonpolar Fraction from Gynostemma pentaphyllum (Thunb.) Makino.

Gynostemma pentaphyllum (Thunb.) Makino (GpM) has been widely used in traditional Chinese medicine (TCM) for the treatment of various diseases including cancer. Most previous studies have focused primarily on polar fractions of GpM for anticancer activities. In this study, a nonpolar fraction EA1.3A from GpM showed potent growth inhibitory activities against four cancer cell lines with IC50 ranging from 31.62 µg/mL to 38.02 µg/mL. Furthermore, EA1.3A also inhibited the growth of breast cancer cell MDA-MB-453 time-dependently, as well as its colony formation ability. EA1.3A induced apoptosis on MDA-MB-453 cells both dose-dependently and time-dependently as analyzed by flow cytometry and verified by western blotting analysis of apoptosis marker cleaved nuclear poly(ADP-ribose) polymerase (cPARP). Additionally, EA1.3A induced cell cycle arrest in G0/G1 phase. Chemical components analysis of EA1.3A by GC-MS revealed that this nonpolar fraction from GpM contains 10 compounds including four alkaloids, three organic esters, two terpenes, and one catechol substance, and all these compounds have not been reported in GpM. In summary, the nonpolar fraction EA1.3A from GpM inhibited cancer cell growth through induction of apoptosis and regulation of cell cycle progression. Our study shed light on new chemical bases for the anticancer activities of GpM and feasibilities to develop new anticancer agents from this widely used medicinal plant.

Nobiletin enhances the efficacy of chemotherapeutic agents in ABCB1 overexpression cancer cells.

Multidrug resistance (MDR) is the major obstacle to the successful chemotherapy treatment of many cancers. Here we found that nobiletin, a citrus methoxyflavone, significantly sensitized ABCB1 overexpressing cells A2780/T and A549/T to chemotherapeutic agents such as paclitaxel (a 433-fold reversal of MDR to PTX at 9 µM), doxorubicin (DOX), docetaxel and dounorubicin. Nobiletin profoundly inhibited ABCB1 transporter activity since it significantly increased the intracellular accumulation of DOX and Flutax-2 in A2780/T cells and decreased the efflux of ABCB1 substrates in Caco2 cells without altering the mRNA and protein expression of ABCB1. Moreover, nobiletin stimulated ATPase activity and inhibited verapamil-stimulated ATPase activity in a concentration-dependent manner, indicating a direct interaction with the transporter. Consistent with these findings, molecular docking analysis also identified favorable binding of nobiletin with the transmemberane region site 1 of homology modeled human ABCB1 transporter. Moreover, the Nrf2 protein expression and phosphorylation levels of AKT/ERK were suppressed by co-treated with nobiletin and PTX at the reversal concentrations, suggesting that inhibition of the AKT/ERK/Nrf2 pathway was associated with the sensitizing effect of nobiletin. These findings encourage further animal and clinical MDR studies with the combination therapy of nobiletin and chemotherapeutic drugs.