Co-treatment With Aripiprazole and Vincristine Sensitizes P-Glycoprotein-Overexpressing Drug-resistant MCF-7/ADR Breast Cancer Cells.

BACKGROUND/AIM: Evidence supports that use of aripiprazole sensitizes drug-resistant oral cancer cells. The aim of the study was to investigate whether aripiprazole can achieve sensitization of highly drug-resistant breast cancer cells, as well as identify its relevant mechanisms of action. MATERIALS AND METHODS: MCF-7/ADR, KB, and KBV20C breast cancer cells were treated with aripiprazole, vincristine (VIC), vinorelbine, vinblastine and their combination. Cell viability assay, annexin V analyses, cellular morphology and density observation with a microscope, western-blotting, fluorescence-activated cell sorting (FACS), and analysis for P-gp inhibitory activity were performed to investigate the drugs' mechanism of action. RESULTS: We found that high drug resistance in MCF-7/ADR cells results from high P-gp inhibitory activity via overexpression of P-gp. Aripiprazole reduced cell viability, increased G2 arrest, and upregulated apoptosis when used as a co-treatment with VIC. Furthermore, we demonstrated that co-treatment with vinorelbine and vinblastine increased the sensitization of MCF-7/ADR breast cancer cells to aripiprazole. We confirmed that VIC-aripiprazole combination has much higher sensitization effects than either VIC-thioridazine or VIC-trifluoperazine co-treatment in MCF-7/ADR cells, since the previously known bipolar drugs (thioridazine and trifluoperazine) has lower P-gp inhibitory activity. However, aripiprazole-induced sensitization was not observed in VIC-treated MDA-MB-231 breast cancer cells suggesting that combination therapy with aripiprazole is specific for P-gp-overexpressing drug-resistant breast cancer cells. CONCLUSION: Co-treatment with low doses of aripiprazole sensitized MCF-7/ADR cells to VIC. Combination therapy with aripiprazole may be a valuable tool for delaying or reducing cancer recurrence by targeting P-gp-overexpressing drug-resistant breast cancer cells.

The influence of cytotoxic drugs on the immunophenotype of blast cells in paediatric B precursor acute lymphoblastic leukaemia.

BACKGROUND: Flow cytometry plays is important in the diagnosis of acute lymphoblastic leukaemia (ALL) and when antigen-specific immunotherapy is indicated. We have investigated the effects of prednisolone, vincristine, daunorubicin, asparaginase and methotrexate on the antigen expression on blast cells that could influence the planning of antigen-specific therapy as well as risk-based treatment assignment. PATIENTS AND METHODS: Patients aged ≤ 17 years with de novo B-cell ALL (B-ALL) were enrolled in the study. Blast cells were isolated and exposed in vitro to 5 individual cytotoxic drugs in logarithmically increasing concentrations. Then, the expression of CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c and CD137 antigens was determined by quantitative flow cytometry. RESULTS: Cytotoxic drugs caused dose-dependent or dose-independent modulation of antigen expression. Daunorubicin caused a dose-dependent down-modulation of CD10, CD19, CD34, CD45 and CD58 and an up-modulation of CD137. Vincristine caused a dose-dependent down-modulation of CD19 and CD58 and an up-modulation of CD45. Daunorubicin also caused dose-independent down-modulation of CD27 and prednisolone down-modulation of CD10, CD19, CD27, CD34 and CD58. Down-modulation of CD20 was detected only in relation to the specific dose of daunorubicin. CONCLUSIONS: The results of the study have shown that cytotoxic drugs can alter the expression of antigens that are important for immunotherapy. Importantly, daunorubicin, prednisolone and vincristine caused down-modulation of CD19 and CD58, suggesting that these drugs are better avoided during bridging therapy prior to bispecific antibodies or CAR-T cell therapy. In addition, immunophenotypic changes on blast cells induced by different drugs could also influence risk-based treatment assignment.

Alendronate-Grafted Nanoemulsions for Bone-Targeted Vincristine Delivery: Preliminary Studies on Cell and Animal Models.

Bone is a site of distant metastases, which are a common cause of morbidity and mortality with a high socio-economic impact, for many malignant tumours. In order to engineer pharmacological therapies that are suitable for this debilitating disease, this experimental work presents injectable lipid nanoemulsions, which are endowed with a long history of safe clinical usage in parenteral nutrition, their loading with vincristine and their grafting with alendronate, with a dual purpose: merging the anticancer activity of bisphosphonates and vincristine, and enhancing bone-targeted delivery. In cell studies, alendronate synergised with the anti-migration activity of vincristine, which is important as migration plays a key role in the metastatisation process. In preliminary animal studies, carried out thanks to IVIS technology, alendronate conjugation enhanced the bone targeting of fluorescently labelled nanoemulsions. These encouraging results will drive further studies on suitable animal models of the disease.

Effect of proteasome inhibitors on canine lymphoma cell response to CHOP chemotherapy in vitro.

The standard treatment for canine lymphoma is the CHOP chemotherapy regimen. Proteasome inhibitors have been employed with CHOP for the treatment of human haematological malignancies but remain to be fully explored in canine lymphoma. We identified an association between poor response to CHOP chemotherapy and high mRNA expression levels of proteasomal subunits in a cohort of 15 canine lymphoma patients, and sought to determine the effect of proteasome inhibitors on the viability of a canine B-cell lymphoma cell line (CLBL-1). The aim of this study was to investigate whether proteasome inhibitors sensitize these cells to the CHOP agents doxorubicin, vincristine and cyclophosphamide (as 4-hydroxycyclophosphamide/4-HC). CLBL-1 cells were sensitive to proteasome inhibition by bortezomib and ixazomib. The IC50 of bortezomib was 15.1 nM and of ixazomib was 59.14 nM. Proteasome inhibitors plus doxorubicin had a synergistic effect on CLBL-1 viability; proteosome inhibitors plus vincristine showed different effects depending on the combination ratio, and there was an antagonistic effect with 4-HC. These results may have clinical utility, as proteasome inhibition could potentially be used with a synergizing CHOP compound to improve responsiveness to chemotherapy for canine lymphoma patients.

Prox1 Suppresses Proliferation and Drug Resistance of Retinoblastoma Cells via Targeting Notch1.

OBJECTIVE: Retinoblastoma (RB) is a prevalent type of eye cancer in youngsters. Prospero homeobox 1 (Prox1) is a homeobox transcriptional repressor and downstream target of the proneural gene that is relevant in lymphatic, hepatocyte, pancreatic, heart, lens, retinal, and cancer cells. The goal of this study was to investigate the role of Prox1 in RB cell proliferation and drug resistance, as well as to explore the underlying Notch1 mechanism. METHODS: Human RB cell lines (SO-RB50 and Y79) and a primary human retinal microvascular endothelial cell line (ACBRI-181) were used in this study. The expression of Prox1 and Notch1 mRNA and protein in RB cells was detected using quantitative real time-polymerase chain reaction (RT-qPCR) and Western blotting. Cell proliferation was assessed after Prox1 overexpression using the Cell Counting Kit-8 and the MTS assay. Drug-resistant cell lines (SO-RB50/vincristine) were generated and treated with Prox1 to investigate the role of Prox1 in drug resistance. We employed pcDNA-Notch1 to overexpress Notch1 to confirm the role of Notch1 in the protective function of Prox1. Finally, a xenograft model was constructed to assess the effect of Prox1 on RB in vivo. RESULTS: Prox1 was significantly downregulated in RB cells. Overexpression of Prox1 effectively decreased RB cell growth while increasing the sensitivity of drug-resistant cells to vincristine. Notch1 was involved in Prox1's regulatory effects. Notch1 was identified as a target gene of Prox1, which was found to be upregulated in RB cells and repressed by increased Prox1 expression. When pcDNA-Notch1 was transfected, the effect of Prox1 overexpression on RB was removed. Furthermore, by downregulating Notch1, Prox1 overexpression slowed tumor development and increased vincristine sensitivity in vivo. CONCLUSION: These data show that Prox1 decreased RB cell proliferation and drug resistance by targeting Notch1, implying that Prox1 could be a potential therapeutic target for RB.

Integrated network pharmacology and metabolomics reveal the action mechanisms of vincristine combined with celastrol against colon cancer.

Colon cancer is associated with a high mortality rate. Vincristine (VCR) is a commonly used chemotherapeutic drug. Celastrol (CEL) is an effective component which exerts inhibitory effects on colon cancer. Combination treatment improves resistance to chemotherapeutic drugs and enhances their efficacy. Therefore, we aimed to explore the molecular mechanisms of VCR combined with CEL in colon cancer treatment. We verified the effects of VCR combined with CEL on the proliferation, cell cycle, and apoptosis of HCT-8 cells. Non-targeted metabolomic techniques were used to analyse the changes in cellular metabolites after administration. Finally, network pharmacology technology was used to screen the potential targets and pathways. VCR combined with CEL had synergistic inhibitory effects on HCT-8 colon cancer cells. Cell metabolomics identified 12 metabolites enriched in metabolic pathways, such as the phenylalanine, tyrosine and tryptophan biosynthesis pathways. Network pharmacology revealed that MAPK1, AKT1, PIK3CB, EGFR, and VEGFA were the key targets. Western blotting revealed that VCR combined with CEL activated the P53 pathway by suppressing the PI3K/AKT signalling pathway activation and Bcl-2 expression, promoting the Bax expression. Therefore, VCR combined with CEL potentially treats colon cancer by increasing the apoptosis, improving energy metabolism, and inhibiting PI3K/AKT pathway in colon cancer cells.

Effects of vincristine and monosodium glutamate on gastrointestinal motility and visceral sensitivity.

BACKGROUND: Chemotherapy-induced adverse effects are an unresolved nightmare. In preclinical studies in rats, the food additive monosodium glutamate (MSG) improved some of the side effects caused by cisplatin, but its effects in other models of chemotherapy-treated animals are not well known. The aim of this study was to test if MSG may improve some of the adverse effects induced by vincristine in rats. METHODS: Young male Wistar rats were exposed or not to MSG (4 g L-1 ) in drinking water from week 0 till 1 week after treatment (week 3). Rats received two cycles of five daily intraperitoneal (ip) injections (Monday to Friday, weeks 1 and 2) of either saline (2 mL kg-1 ) or vincristine (0.1 mg kg-1 ). Gastrointestinal motility was measured in vivo by radiological methods after the first and tenth ip administrations. On week 3, the threshold for mechanical somatic and colorectal sensitivity was recorded using Von Frey filaments applied to the paws and an intracolonic balloon, respectively. Finally, samples of the terminal ileum and distal colon were histologically evaluated in sections. KEY RESULTS: Vincristine reduced body weight gain, food intake, and upper gastrointestinal transit, caused somatic (but not visceral) hypersensitivity and increased the thickness of the submucosal and muscle layers of the small intestine. In vincristine-treated animals, MSG partially prevented gastrointestinal dysmotility and reduced visceral sensitivity but did not improve structural alterations of the small intestine. CONCLUSIONS & INFERENCES: MSG could be used as an adjuvant to conventional treatments to improve some gastrointestinal dysfunctions caused by chemotherapy.

Enhanced anti-tumor activity of transferrin/folate dual-targeting magnetic nanoparticles using chemo-thermo therapy on retinoblastoma cancer cells Y79.

Malignant neoplasms are one of the main causes of death, especially in children, on a global scale, despite strenuous efforts made at advancing both diagnostic and therapeutic modalities. In this regard, a new nanocarrier Vincristine (VCR)-loaded Pluronic f127 polymer-coated magnetic nanoparticles conjugated with folic acid and transferrin (PMNP-VCR-FA-TF) were synthesized and characterized by various methods. The cytotoxicity of these nanoparticles was evaluated in vitro and ex vivo conditions. The in vitro anti-tumor effect of the nanoparticles was evaluated by colony formation assay (CFA) and reactive oxygen species (ROS) in Y79 cell line. The results showed that nanoparticles with two ligands conferred greater toxicity toward Y79 cancer cells than ARPE19 normal cells. Under an alternating magnetic field (AMF), these nanoparticles demonstrated a high specific absorption rate. The CFA and ROS results indicated that the AMF in combination with PMNP-VCR-FA-TF conferred the highest cytotoxicity toward Y79 cells compared with other groups (P < 0.05). PMNP-VCR-FA-TF could play an important role in converting externally applied radiofrequency energy into heat in cancer cells. The present study confirmed that dual targeting chemo-hyperthermia using PMNP-VCR-FA-TF was significantly more effective than hyperthermia or chemotherapy alone, providing a promising platform for precision drug delivery as an essential component in the chemotherapy of retinoblastoma.

Engineering redirected NF-κB/OIP5 expression programs to enhance tumor responses to chemotherapy in bladder cancer.

Nuclear factor kappa-B (NF-κB), a pivotal transcriptional regulator, plays a crucial role in modulating downstream genes implicated in tumor drug resistance. We establish a programmable system within bladder cancer cells to tailor drug responses by employing a synthetic clustered regularly interspaced short palindromic repeats (CRISPR)-based expression strategy that emulates natural transcriptional regulators. Our investigation uncovers the functional significance of Opa-interacting protein 5 (OIP5), upregulated upon NF-κB activation, as a key regulator governing drug-resistance to vincristine (VCR) treatment in bladder cancer. Through engineered guide RNAs (sgRNAs) targeting OIP5 to integrate NF-κB aptamers, we construct a modular scaffold RNA that encodes both the target locus and regulatory functionality. This engineered CRISPR scaffold RNA effectively responds to VCR stimulus by binding with activated NF-κB. Intriguingly, it redirects NF-κB to attenuate OIP5 expression-a reversal of its original role-while concurrently obstructing multiple NF-κB-mediated drug resistance pathways. This dual action thwarts drug resistance development. Further enhancing therapeutic potential, we develop a versatile nanoparticle system capable of co-delivering CRISPR scaffold RNAs and VCR. This synergistic approach demonstrates potent anti-tumor effects in both in vitro and in vivo settings. Our nanoparticle-mediated combination presents a compelling proof-of-concept, showcasing the utility of engineered CRISPR-based synthetic expression programs to reconfigure cellular drug responses and heighten tumor cell susceptibility to chemotherapy.

TCFL5 knockdown sensitizes DLBCL to doxorubicin treatment via regulation of GPX4.

BACKGROUND: Resistance to chemo-drug is a major cause of bad outcome in diffuse large B-cell lymphoma (DLBCL). It was reported that TCFL5 may be related to chemoresistance in childhood acute lymphoblastic leukemia. However, it is still unclear whether TCFL5 is involved in DLBCL drug-resistance. METHODS: To explore the underlying mechanism of doxorubicin resistance, recombinant lentivirus was applied to control expression of TCFL5 in DLBCL cells. CCK-8 assay was perfomed to investigate the influence of doxorubicin on proliferation of TCFL5-overexpressed or sh-TCFL5 DLBCL cells. Correlation between TCFL5 and GPX4 was analyzed with bioinformatic methods, which was further confirmed by qPCR and western blot. TCFL5 overexpression conferred doxorubicin resistance via regulating GPX4 and was verified by TUNEL assay and western blot in vitro and mice model in vivo. RESULTS: TCFL5 was enriched in DLBCL cells and conferred doxorubicin resistance through binding to GPX4. Inhibition of TCFL5 enhanced the sensitivity of DLBCL cells to doxorubicin. GPX4 knockdown reversed doxorubicin resistance in TCFL5-overexpressed DLBCL cells. CONCLUSION: DLBCL cells overexpress TCFL5 that promotes chemoresistance by regulating GPX4. Targeting TCFL5 may provide a prospective therapeutic strategy for doxorubicin-resistant DLBCL.

Cytotoxicity of alkaline serine protease (ASPNJ) on Jurkat cells and its correlation with changes in the expression of membrane-associated proteins.

We aim to study the inhibitory effect of alkaline serine protease (ASPNJ) on lymphocytic leukemia Jurkat cells and its related mechanism through examining the expression of membrane proteins or membrane-associated proteins. MTT assay and trypan blue staining were used to detect the inhibitory effect of ASPNJ on the proliferation and growth of Jurkat cells. Wright-Giemsa staining was used to observe the effect of ASPNJ on the morphology of Jurkat cells. The effect of ASPNJ on Jurkat cell apoptosis was detected by flow cytometry. Two-dimensional electrophoresis-mass spectrometry (2-DE-MS) was used to detect and identify the differentially expressed proteins of Jurkat cells treated with ASPNJ (4 µg/mL, 3 h), of which three were selected and verified by Western blot. ASPNJ significantly inhibited the proliferation of leukemia cells (Raji, U937, and Jurkat), caused obvious morphological changes, and induced apoptosis of Jurkat cells. ASPNJ also increased the sensitivity of Jurkat cells to vincristine (VCR). Seven differentially expressed proteins were obtained through 2DE-MS, of which Peroxiredoxin-6 (PRDX6), Calcium-binding protein (CHP1), and 40S ribosomal protein SA (RPSA) were validated. ASPNJ can cause significant toxic effects on Jurkat cells and enhance the effects of VCR. The mechanism of action of ASPNJ on Jurkat cells may be related to differentially expressed proteins such as PRDX6. This study provides a new experimental basis and direction for antileukemia research.

Genome-wide CRISPR screening uncovers potential targets and mechanisms of vincristine resistance in DLBCL.

In this issue, Rovsing et al. employ unbiased genome-wide CRISPR screening and functional cellular assays to investigate the cellular response to vincristine, an important component of the front-line DLBCL treatment R-CHOP. Their findings reveal intriguing targets and mechanisms that hold promise for enhancing DLBCL treatment and provide a foundation for the development of future drug regimens. This research prompts further exploration of the translational potential to advance more effective and individualized approaches in the clinical management of DLBCL. Commentary on: Rovsing et al. Resistance to vincristine in DLBCL by disruption of p53-induced cell cycle arrest and apoptosis mediated by KIF18B and USP28. Br J Haematol 2023;202:825-839.

Vincristine Enhances the Efficacy of MEK Inhibitors in Preclinical Models of KRAS-mutant Colorectal Cancer.

Mutations in KRAS are found in more than 50% of tumors from patients with metastatic colorectal cancer (mCRC). However, direct targeting of most KRAS mutations is difficult; even the recently developed KRASG12C inhibitors failed to show significant benefit in patients with mCRC. Single agents targeting mitogen-activated protein kinase kinase (MEK), a downstream mediator of RAS, have also been ineffective in colorectal cancer. To identify drugs that can enhance the efficacy of MEK inhibitors, we performed unbiased high-throughput screening using colorectal cancer spheroids. We used trametinib as the anchor drug and examined combinations of trametinib with the NCI-approved Oncology Library version 5. The initial screen, and following focused validation screens, identified vincristine as being strongly synergistic with trametinib. In vitro, the combination strongly inhibited cell growth, reduced clonogenic survival, and enhanced apoptosis compared with monotherapies in multiple KRAS-mutant colorectal cancer cell lines. Furthermore, this combination significantly inhibited tumor growth, reduced cell proliferation, and increased apoptosis in multiple KRAS-mutant patient-derived xenograft mouse models. In vivo studies using drug doses that reflect clinically achievable doses demonstrated that the combination was well tolerated by mice. We further determined that the mechanism underlying the synergistic effect of the combination was due to enhanced intracellular accumulation of vincristine associated with MEK inhibition. The combination also significantly decreased p-mTOR levels in vitro, indicating that it inhibits both RAS-RAF-MEK and PI3K-AKT-mTOR survival pathways. Our data thus provide strong evidence that the combination of trametinib and vincristine represents a novel therapeutic option to be studied in clinical trials for patients with KRAS-mutant mCRC. SIGNIFICANCE: Our unbiased preclinical studies have identified vincristine as an effective combination partner for the MEK inhibitor trametinib and provide a novel therapeutic option to be studied in patients with KRAS-mutant colorectal cancer.

Vincristine-associated total antioxidant and oxidant status of ovaries and in vitro nuclear oocyte maturation in dogs with canine transmissible venereal tumor.

The aim of this study is to evaluate the effects of scheduled vincristine sulfate therapy on canine oocyte quality and nuclear oocyte maturation, associated with total antioxidant and oxidant status of ovaries and Anti-Müllerian Hormone (AMH) concentrations in dogs with Canine Transmissible Venereal Tumor (CTVT). Six bitches suffering from CTVT and six healthy bitches were included in the study. Hemogram was carried out weekly. AMH measurements and ovariohysterectomy operations were performed after the termination of vincristine sulfate therapies. Tissue samples from ovaries were utilized for Malondialdehyde (MDA), reduced Glutathione (GSH), Superoxide Dismutase (SOD), Total Anti-oxidative Status (TAS), Total Oxidative Status (TOS) measurements, and Oxidative Stress Index (OSI) was calculated. Collected oocytes were evaluated for meiotic competence, after In Vitro Maturation (IVM) and parthenogenetic activation. No difference between the two groups was observed in hematologic parameters (P > 0.05). Meiotic stages of Germinal Vesicle Break Down (GVBD), Metaphase I (MI), and Metaphase II (MII) were significantly different between groups (P < 0.05). The number of oocytes reaching MII and meiotic resumption was lower in the CTVT group. Furthermore, AMH concentrations, oxidant parameters (OSI, TOS, and MDA), and antioxidant parameters (GSH, SOD, and TAS) were also statistically different between groups (P < 0.05). The results of this study show that vincristine sulfate application in the treatment of CTVT could alter oxidant/antioxidant status in ovaries. Apart from these, oocyte quality and IVM rates seem to decline related to gonadotoxicity. Moreover, AMH could be an important marker in the evaluation of oocyte qualities in bitches, as it is in women.

Resistance to vincristine in DLBCL by disruption of p53-induced cell cycle arrest and apoptosis mediated by KIF18B and USP28.

The frontline therapy R-CHOP for patients with diffuse large B-cell lymphoma (DLBCL) has remained unchanged for two decades despite numerous Phase III clinical trials investigating new alternatives. Multiple large studies have uncovered genetic subtypes of DLBCL enabling a targeted approach. To further pave the way for precision oncology, we perform genome-wide CRISPR screening to uncover the cellular response to one of the components of R-CHOP, vincristine, in the DLBCL cell line SU-DHL-5. We discover important pathways and subnetworks using gene-set enrichment analysis and protein-protein interaction networks and identify genes related to mitotic spindle organization that are essential during vincristine treatment. The inhibition of KIF18A, a mediator of chromosome alignment, using the small molecule inhibitor BTB-1 causes complete cell death in a synergistic manner when administered together with vincristine. We also identify the genes KIF18B and USP28 of which CRISPR/Cas9-directed knockout induces vincristine resistance across two DLBCL cell lines. Mechanistic studies show that lack of KIF18B or USP28 counteracts a vincristine-induced p53 response suggesting that resistance to vincristine has origin in the mitotic surveillance pathway (USP28-53BP1-p53). Collectively, our CRISPR screening data uncover potential drug targets and mechanisms behind vincristine resistance, which may support the development of future drug regimens.

Enhancing the Impact of Chemotherapy on Ewing Sarcoma Cells through Combination with Cold Physical Plasma.

Although Ewing's sarcoma (ES) is a rare, but very aggressive tumor disease affecting the musculoskeletal system, especially in children, it is very aggressive and difficult to treat. Although medical advances and the establishment of chemotherapy represent a turning point in the treatment of ES, resistance to chemotherapy, and its side effects, continue to be problems. New treatment methods such as the application of cold physical plasma (CPP) are considered potential supporting tools since CPP is an exogenous source of reactive oxygen and nitrogen species, which have similar mechanisms of action in the tumor cells as chemotherapy. This study aims to investigate the synergistic effects of CPP and commonly used cytostatic chemotherapeutics on ES cells. The chemotherapy drugs doxorubicin and vincristine, the most commonly used in the treatment of ES, were applied to two different ES cell lines (RD-ES and A673) and their IC20 and IC50 were determined. In addition, individual chemotherapeutics in combination with CPP were applied to the ES cells and the effects on cell growth, cell viability, and apoptosis processes were examined. A single CPP treatment resulted in the dose-dependent growth inhibition of ES cells. The combination of different cytostatics and CPP led to significant growth inhibition, a reduction in cell viability, and higher rates of apoptosis compared to cells not additionally exposed to CPP. The combination of CPP treatment and the application of cytostatic drugs to ES cells showed promising results, significantly enhancing the cytotoxic effects of chemotherapeutic agents. These preclinical in vitro data indicate that the use of CPP can enhance the efficacy of common cytostatic chemotherapeutics, and thus support the translation of CPP as an anti-tumor therapy in clinical routine.

Design, synthesis and biological evaluation of matrine-dithiocarbamate hybrids as potential anticancer agents.

A series of novel matrine-dithiocarbamate (DTC) hybrids were efficiently synthesised from matrine through a three-step sequence involving basic hydrolysis, esterification, and DTC formation. They were evaluated for their in vitro cytotoxic potency on several human cancer and normal cells. All matrine-DTC hybrids were much more toxic against the human hepatoma cell line HepG2 than the parent matrine. Hybrid 4l (IC50 = 31.39 µM) was the most potent compound against HepG2 cells, being 156- and 3-fold more toxic than matrine (IC50 > 4900 µM) and the reference vincristine (VCR, IC50 = 93.67 µM), respectively. Moreover, hybrid 4l was less toxic to normal human embryonic kidney cell line HEK-293T, with a higher selectivity index (SI, HEK-293T/HepG2 ≈ 6) than matrine (SI ≈ 1) and VCR (SI ≈ 1). The structure-activity relationship analysis indicated that selectivity was greatly boosted when 4-(trifluoromethyl)benzyl was incorporated into the hybrids (4f and 4l). In addition, the hybrid 4l was also highly toxic to the other five types of human cancer cells (Calu-1, SK-BR-3, HUH-7, 786-O and SK-OV-3; IC50 = 44.18-112.19 µM), whereas it was relatively less toxic to the corresponding normal cells (WI-38, LX-2, HEK-293T and KGN; IC50 = 81.48-195.17 µM). Further mechanistic studies showed that hybrid 4l induced apoptosis in a concentration-dependent manner in HepG2 cells. Our results demonstrate that the cytotoxic activity of matrine can be highly enhanced by hybridisation with DTC. Hybrid 4l has promising applications in anticancer drug development.

Potentiating Biosynthesis of Alkaloids and Polyphenolic Substances in Catharanthus roseus Plant Using ĸ-Carrageenan.

Catharanthus roseus is a medicinal plant that produces indole alkaloids, which are utilized in anticancer therapy. Vinblastine and vincristine, two commercially important antineoplastic alkaloids, are mostly found in the leaves of Catharanthus roseus. ĸ-carrageenan has been proven as plant growth promoting substance for a number of medicinal and agricultural plants. Considering the importance of ĸ-carrageenan as a promoter of plant growth and phytochemical constituents, especially alkaloids production in Catharanthus roseus, an experiment was carried out to explore the effect of ĸ-carrageenan on the plant growth, phytochemicals content, pigments content, and production of antitumor alkaloids in Catharanthus roseus after planting. Foliar application of ĸ-carrageenan (at 0, 400, 600 and 800 ppm) significantly improved the performance of Catharanthus roseus. Phytochemical analysis involved determining the amount of total phenolics (TP), flavonoids (F), free amino acids (FAA), alkaloids (TAC) and pigments contents by spectrophotometer, minerals by ICP, amino acids, phenolic compounds and alkaloids (Vincamine, Catharanthine, Vincracine (Vincristine), and vinblastine) analysis uses HPLC. The results indicated that all examined ĸ-carrageenan treatments led to a significant (p ≤ 0.05) increase in growth parameters compared to the untreated plants. Phytochemical examination indicates that the spray of ĸ-carrageenan at 800 mg L-1 increased the yield of alkaloids (Vincamine, Catharanthine and Vincracine (Vincristine)) by 41.85 µg/g DW, total phenolic compounds by 3948.6 µg gallic/g FW, the content of flavonoids 951.3 µg quercetin /g FW and carotenoids content 32.97 mg/g FW as compared to the control. An amount of 400 ppm ĸ-carrageenan treatment gave the best contents of FAA, Chl a, Chl b and anthocyanin. The element content of K, Ca, Cu, Zn and Se increased by treatments. Amino acids constituents and phenolics compounds contents were altered by ĸ-carrageenan.

Neuroprotective mechanism of Ajugarin-I against Vincristine-Induced neuropathic pain via regulation of Nrf2/NF-κB and Bcl2 signalling.

Vincristine (VCR) is a well-known chemotherapeutic agent that frequently triggers neuropathic pain. Ajugarin-I (Aju-I) isolated from Ajuga bracteosa exerts antioxidant, anti-inflammatory, and neuroprotective properties. The present study was designed to investigate the ameliorative potential of Aju-I against VCR-induced neuropathic pain and explored the underlying mechanism involved. The neuroprotective potential of Aju-I was first confirmed against hydrogen peroxide (H2O2)-induced cytotoxicity and oxidative stress in PC12 cells. For neuropathic pain induction, vincristine was given intraperitoneally (i.p.) into adult male albino mice (BALB/c) of the same age (8-12 weeks old) for 10 days (days 1-10). Aju-I (1 and 5 mg/kg) doses were administered from day 11 to 21 intraperitoneally (i.p.) after the neuropathic induction. Initially, behavioral tests such as thermal hyperalgesia, mechanical allodynia, and cold allodynia were performed to investigate the antinociceptive potential of Ajugarin-I (1 and 5 mg/kg, b.w). The nuclear factor-erythroid factor 2-related factor 2(Nrf2), nuclear factor-κB (NF-κB), BCL2-associated × protein (Bax), and B-cell-lymphoma-2 (Bcl-2) signaling proteins were determined by immunohistochemistry and western blot. Additionally, inflammatory cytokines, antioxidant, and oxidative stress parameters were also measured in the spinal cord and sciatic nerve. The behavioral results demonstrated that Aju-I (5 mg/kg) markedly alleviated VCR-induced neuropathic pain behaviors including hyperalgesia and allodynia. It reversed the histological alterations caused by VCR in the sciatic nerve, spinal cord, and brain. It significantly alleviated oxidative stress and inflammation by regulating the immunoreactivity of Nrf2/NF-κB signaling. It suppressed apoptosis by regulating the immunoreactivity of Bcl-2/Bax and Caspase-3. The flow cytometry and comet analysis also confirmed its anti-apoptotic potential. It considerably improved the antioxidant status and mitigated VCR-induced inflammatory cytokines. High-performance liquid chromatography (HPLC) analysis indicated that Aju-I crosses the blood-brain barrier (BBB) and penetrated the brain tissue. These findings suggest that Aju-I treatment inhibited vincristine-induced neuropathy via regulation of Nrf2/NF-κB and Bcl2 signaling.

Simplified Derivatives of Tetrandrine as Potent and Specific P-gp Inhibitors to Reverse Multidrug Resistance in Cancer Chemotherapy.

Targeted inhibition of a drug efflux transporter P-glycoprotein (P-gp) is an important strategy to reverse multidrug resistance in cancer chemotherapy. In this study, a rationally structural simplification to natural tetrandrine was performed based on molecular dynamics simulation and fragment growth, leading to an easily prepared, novel, and simplified compound OY-101 with high reversal activity and low cytotoxicity. Its excellent synergistic anti-cancer effect with vincristine (VCR) against drug-resistant cells Eca109/VCR was confirmed by reversal activity assay, flow cytometry, plate clone formation assay, and drug synergism analysis (IC50 = 9.9 nM, RF = 690). Further mechanism study confirmed that the OY-101 was a specific and efficient P-gp inhibitor. Importantly, OY-101 increased VCR sensitization in vivo without obvious toxicity. Overall, our findings may provide an alternative strategy for the design of novel specific P-gp inhibitor as an anti-tumor chemotherapy sensitizer.