2-Methoxyestradiol, an Endogenous 17ß-Estradiol Metabolite, Induces Antimitogenic and Apoptotic Actions in Oligodendroglial Precursor Cells and Triggers Endoreduplication via the p53 Pathway.

The abnormal growth of oligodendrocyte precursor cells (OPCs) significantly contributes to the progression of glioblastoma tumors. Hence, molecules that block OPC growth may be of therapeutic importance in treating gliomas. 2-Methoxyestradiol (2ME), an endogenous tubulin-interacting metabolite of estradiol, is effective against multiple proliferative disorders. Based on its anti-carcinogenic and anti-angiogenic actions, it is undergoing phase II clinical trials. We hypothesize that 2ME may prevent glioma growth by targeting OPC growth. Here, we tested this hypothesis by assessing the impact of 2ME on the growth of an OPC line, "Oli-neu", and dissected the underlying mechanism(s). Treatment with 2ME inhibited OPC growth in a concentration-dependent manner, accompanied by significant upregulation in the expression of p21 and p27, which are negative cell-cycle regulators. Moreover, treatment with 2ME altered OPC morphology from multi-arm processes to rounded cells. At concentrations of 1uM and greater, 2ME induced apoptosis, with increased expressions of caspase 3, PARP, and caspase-7 fragments, externalized phosphatidylserine staining/APOPercentage, and increased mitochondrial activity. Flow cytometry and microscopic analysis demonstrated that 2ME triggers endoreduplication in a concentration-dependent fashion. Importantly, 2ME induced cyclin E, JNK1/2, and p53 expression, as well as OPC fusion, which are key mechanisms driving endoreduplication and whole-genome duplication. Importantly, the inhibition of p53 with pifithrin-α rescued 2ME-induced endoreduplication. The pro-apoptotic and endoreduplication actions of 2ME were accompanied by the upregulation of survivin, cyclin A, Cyclin B, Cyclin D2, and ppRB. Similar growth inhibitory, apoptotic, and endoreduplication effects of 2ME were observed in CG4 cells. Taken together, our findings provide evidence that 2ME not only inhibits OPC growth and triggers apoptosis, but also activates OPCs into survival (fight or flight) mode, leading to endoreduplication. This inherent survival characteristic of OPCs may, in part, be responsible for drug resistance in gliomas, as observed for many tubulin-interacting drugs. Importantly, the fate of OPCs after 2ME treatment may depend on the cell-cycle status of individual cells. Combining tubulin-interfering molecules with drugs such as pifithrin-α that inhibit endoreduplication may help inhibit OPC/glioma growth and limit drug resistance.

Next generation antimitotic ß-carboline derivatives modulate microtubule dynamics and downregulate NF-κB, ERK 1/2 and phospho HSP 27.

AIM: Exploring the efficacy of ß-carboline-based molecular inhibitors in targeting microtubules for the development of novel anticancer therapeutics. MATERIALS AND METHODS: We synthesized a series of 1-Aryl-N-substituted-ß-carboline-3-carboxamide compounds and evaluated their cytotoxicity against human lung carcinoma (A549) cells using the MTT assay. Normal lung fibroblast cells (WI-38) were used to assess compound selectivity. The mechanism of action of MJ-211 was elucidated through Western blot analysis of key pro-apoptotic and cell cycle regulatory proteins. Additionally, the inhibitory effect of MJ-211 on multicellular 3D spheroid growth of A549 cells was evaluated. KEY FINDINGS: Lead compound MJ-211 exhibited remarkable cytotoxicity against A549 cells with an IC50 of 4.075 µM at 24 h treatment and IC50 of 1.7 nM after 72 h of treatment, while demonstrating selectivity towards normal WI-38 cells. MJ-211 activated pro-apoptotic factors Bim and p53, and suppressed Cyclin B1, Phospho HSP 27, BubR1, Mad 2, ERK1/2, and NF-κB, indicating its potent antimitotic and pro-apoptotic effects. MJ-211 significantly suppressed the migration of cells and inhibited the growth of A549 cell-derived multicellular 3D spheroids, highlighting its efficacy in a more physiologically relevant model. SIGNIFICANCE: Cytotoxic effect of MJ-211 against cancer cells, selectivity towards normal cells, and ability to modulate key regulatory proteins involved in apoptosis and cell cycle progression underscore its potential as a promising template for further anticancer lead optimization. Moreover, the inhibitory effect of MJ-211 on multicellular spheroid growth suggests its efficacy in combating tumor heterogeneity and resistance mechanisms, thereby offering a promising avenue for future anticancer drug development.

Modification of the phenyl ring B of phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates by pyridinyl moiety leads to novel antimitotics targeting the colchicine-binding site.

A series of 8 novel pyridinyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PYRIB-SOs) were designed, prepared and evaluated for their mechanism of action. PYRIB-SOs were found to have antiproliferative activity in the nanomolar to submicromolar range on several breast cancer cell lines. Moreover, subsequent biofunctional assays indicated that the most potent PYRIB-SOs 1-3 act as antimitotics binding to the colchicine-binding site (C-BS) of α, ß-tubulin and that they arrest the cell cycle progression in the G2/M phase. Microtubule immunofluorescence and tubulin polymerisation assay confirm that they disrupt the cytoskeleton through inhibition of tubulin polymerisation as observed with microtubule-destabilising agents. They also show good overall theoretical physicochemical, pharmacokinetic and druglike properties. Overall, these results show that PYRIB-SOs is a new family of promising antimitotics to be further studied in vivo for biopharmaceutical and pharmacodynamic evaluations.

Control of cell proliferation by memories of mitosis.

Mitotic duration is tightly constrained, and extended mitosis is characteristic of problematic cells prone to chromosome missegregation and genomic instability. We show here that mitotic extension leads to the formation of p53-binding protein 1 (53BP1)-ubiquitin-specific protease 28 (USP28)-p53 protein complexes that are transmitted to, and stably retained by, daughter cells. Complexes assembled through a Polo-like kinase 1-dependent mechanism during extended mitosis and elicited a p53 response in G1 that prevented the proliferation of the progeny of cells that experienced an approximately threefold extended mitosis or successive less extended mitoses. The ability to monitor mitotic extension was lost in p53-mutant cancers and some p53-wild-type (p53-WT) cancers, consistent with classification of TP53BP1 and USP28 as tumor suppressors. Cancers retaining the ability to monitor mitotic extension exhibited sensitivity to antimitotic agents.

BP-M345 as a Basis for the Discovery of New Diarylpentanoids with Promising Antimitotic Activity.

Recently, the diarylpentanoid BP-M345 (5) has been identified as a potent in vitro growth inhibitor of cancer cells, with a GI50 value between 0.17 and 0.45 µM, showing low toxicity in non-tumor cells. BP-M345 (5) promotes mitotic arrest by interfering with mitotic spindle assembly, leading to apoptotic cell death. Following on from our previous work, we designed and synthesized a library of BP-M345 (5) analogs and evaluated the cell growth inhibitory activity of three human cancer cell lines within this library in order to perform structure-activity relationship (SAR) studies and to obtain compounds with improved antimitotic effects. Four compounds (7, 9, 13, and 16) were active, and the growth inhibition effects of compounds 7, 13, and 16 were associated with a pronounced arrest in mitosis. These compounds exhibited a similar or even higher mitotic index than BP-M345 (5), with compound 13 displaying the highest antimitotic activity, associated with the interference with mitotic spindle dynamics, inducing spindle collapse and, consequently, prolonged mitotic arrest, culminating in massive cancer cell death by apoptosis.

Evaluation of ABT-751, a novel anti-mitotic agent able to overcome multi-drug resistance, in melanoma cells.

PURPOSE: Drug efflux transporter associated multi-drug resistance (MDR) is a potential limitation in the use of taxane chemotherapies for the treatment of metastatic melanoma. ABT-751 is an orally bioavailable microtubule-binding agent capable of overcoming MDR and proposed as an alternative to taxane-based therapies. METHODS: This study compares ABT-751 to taxanes in vitro, utilizing seven melanoma cell line models, publicly available gene expression and drug sensitivity databases, a lung cancer cell line model of MDR drug efflux transporter overexpression (DLKP-A), and drug efflux transporter ATPase assays. RESULTS: Melanoma cell lines exhibit a low but variable protein and RNA expression of drug efflux transporters P-gp, BCRP, and MDR3. Expression of P-gp and MDR3 correlates with sensitivity to taxanes, but not to ABT-751. The anti-proliferative IC50 profile of ABT-751 was higher than the taxanes docetaxel and paclitaxel in the melanoma cell line panel, but fell within clinically achievable parameters. ABT-751 IC50 was not impacted by P-gp-overexpression in DKLP-A cells, which display strong resistance to the P-gp substrate taxanes compared to DLKP parental controls. The addition of ABT-751 to paclitaxel treatment significantly decreased cell proliferation, suggesting some reversal of MDR. ATPase activity assays suggest that ABT-751 is a potential BCRP substrate, with the ability to inhibit P-gp ATPase activity. CONCLUSION: Our study confirms that ABT-751 is active against melanoma cell lines and models of MDR at physiologically relevant concentrations, it inhibits P-gp ATPase activity, and it may be a BCRP and/or MDR3 substrate. ABT-751 warrants further investigation alone or in tandem with other drug efflux transporter inhibitors for hard-to-treat MDR melanoma.

Amelioration of Tumor-promoting Microenvironment via Vascular Remodeling and CAF Suppression Using E7130: Biomarker Analysis by Multimodal Imaging Modalities.

E7130 is a novel anticancer agent created from total synthetic study of the natural compound norhalichondrin B. In addition to inhibiting microtubule dynamics, E7130 also ameliorates tumor-promoting aspects of the tumor microenvironment (TME) by suppressing cancer-associated fibroblasts (CAF) and promoting remodeling of tumor vasculature. Here, we demonstrate TME amelioration by E7130 using multi-imaging modalities, including multiplexed mass cytometry [cytometry by time-of-flight (CyTOF)] analysis, multiplex IHC analysis, and MRI. Experimental solid tumors characterized by large numbers of CAFs in TME were treated with E7130. E7130 suppressed LAP-TGFß1 production, a precursor of TGFß1, in CAFs but not in cancer cells; an effect that was accompanied by a reduction of circulating TGFß1 in plasma. To our best knowledge, this is the first report to show a reduction of TGFß1 production in TME. Furthermore, multiplex IHC analysis revealed reduced cellularity and increased TUNEL-positive apoptotic cells in E7130-treated xenografts. Increased microvessel density (MVD) and collagen IV (Col IV), an extracellular matrix (ECM) component associated with endothelial cells, were also observed in the TME, and plasma Col IV levels were also increased by E7130 treatment. MRI revealed increased accumulation of a contrast agent in xenografts. Moreover, diffusion-weighted MRI after E7130 treatment indicated reduction of tumor cellularity and interstitial fluid pressure. Overall, our findings strongly support the mechanism of action that E7130 alters the TME in therapeutically beneficial ways. Importantly, from a translational perspective, our data demonstrated MRI as a noninvasive biomarker to detect TME amelioration by E7130, supported by consistent changes in plasma biomarkers.

Frentizole, a Nontoxic Immunosuppressive Drug, and Its Analogs Display Antitumor Activity via Tubulin Inhibition.

Antimitotic agents are one of the more successful types of anticancer drugs, but they suffer from toxicity and resistance. The application of approved drugs to new indications (i.e., drug repurposing) is a promising strategy for the development of new drugs. It relies on finding pattern similarities: drug effects to other drugs or conditions, similar toxicities, or structural similarity. Here, we recursively searched a database of approved drugs for structural similarity to several antimitotic agents binding to a specific site of tubulin, with the expectation of finding structures that could fit in it. These searches repeatedly retrieved frentizole, an approved nontoxic anti-inflammatory drug, thus indicating that it might behave as an antimitotic drug devoid of the undesired toxic effects. We also show that the usual repurposing approach to searching for targets of frentizole failed in most cases to find such a relationship. We synthesized frentizole and a series of analogs to assay them as antimitotic agents and found antiproliferative activity against HeLa tumor cells, inhibition of microtubule formation within cells, and arrest at the G2/M phases of the cell cycle, phenotypes that agree with binding to tubulin as the mechanism of action. The docking studies suggest binding at the colchicine site in different modes. These results support the repurposing of frentizole for cancer treatment, especially for glioblastoma.

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamide salts: Novel hydrosoluble prodrugs of antimitotics selectively bioactivated by the cytochrome P450 1A1 in breast cancer cells.

4-(3-Alkyl-2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamides (PAIB-SAs) are members of a new family of prodrugs bioactivated by cytochrome P450 1A1 (CYP1A1) in breast cancer cells into their potent 4-(2-oxoimidazolidin-1-yl)-N-phenylbenzenesulfonamide metabolites (PIB-SAs). One of the predominant problems for the galenic formulation and administration of PAIB-SAs in animal studies is their poor hydrosolubility. To circumvent that difficulty, we report the design, the synthesis, the chemical characterization, the evaluation of the aqueous solubility, the antiproliferative activity and the mechanism of action of 18 new Na+, K+ and Li+ salts of PAIB-SAs. Our results evidenced that the latter exhibited highly selective antiproliferative activity toward MCF7 and MDA-MB-468 breast cancer cells expressing endogenously CYP1A1 compared to insensitive MDA-MB-231 and HaCaT cells. Moreover, PAIB-SA salts 1-18 are significantly more hydrosoluble (3.9-9.4 mg/mL) than their neutral counterparts (< 0.0001 mg/mL). In addition, the most potent PAIB-SA salts 1-3 and 10-12 arrested the cell cycle progression in the G2/M phase and disrupted the cytoskeleton's dynamic assembly. Finally, PAIB-SA salts are N-dealkylated by CYP1A1 into their corresponding PIB-SA metabolites, which are potent antimitotics. In summary, our results show that our water-soluble PAIB-SA salts, notably the sodium salts, still exhibit potent antiproliferative efficacy and remain prone to CYP1A1 bioactivation. In addition, these PAIB-SA salts will allow the development of galenic formulations suitable for further biopharmaceutical and pharmacodynamic studies.

Organometallic Ru, Os, Rh and Ir half-sandwich conjugates of ispinesib - impact of the organometallic group on the antimitotic activity.

Antimitotic agents are among the most important drugs used in anticancer therapy. Kinesin spindle protein (KSP) was proposed as a promising target for new antimitotic drugs. Herein, we report the synthesis of Ru, Os, Rh, and Ir half-sandwich complexes with the KSP inhibitor ispinesib and its (S)-enantiomer. Conjugation of the organometallic moiety with ispinesib and its (S)-enantiomer resulted in a significantly increased cytotoxicity of up to 5.6-fold compared to the parent compounds, with IC50 values in the nanomolar range. The most active derivatives were the ispinesib Ru and Rh conjugates which were able to generate reactive oxygen species (ROS), which may at least partially explain their high cytotoxicity. At the same time, the Os and Ir derivatives acted as KSP inhibitors with no effects on ROS generation.

ER-851, a Novel Selective Inhibitor of AXL, Overcomes Resistance to Antimitotic Drugs.

Innate and adaptive resistance to cancer therapies, such as chemotherapies, molecularly targeted therapies, and immune-modulating therapies, is a major issue in clinical practice. Subpopulations of tumor cells expressing the receptor tyrosine kinase AXL become enriched after treatment with antimitotic drugs, causing tumor relapse. Elevated AXL expression is closely associated with drug resistance in clinical samples, suggesting that AXL plays a pivotal role in drug resistance. Although several molecules with AXL inhibitory activity have been developed, none have sufficient activity and selectivity to be clinically effective when administered in combination with a cancer therapy. Here, we report a novel small molecule, ER-851, which is a potent and highly selective AXL inhibitor. To investigate resistance mechanisms and identify driving molecules, we conducted a comprehensive gene expression analysis of chemoresistant tumor cells in mouse xenograft models of genetically engineered human lung cancer and human triple-negative breast cancer. Consistent with the effect of AXL knockdown, cotreatment of ER-851 and antimitotic drugs produced an antitumor effect and prolonged relapse-free survival in the mouse xenograft model of human triple-negative breast cancer. Importantly, when orally administered to BALB/c mice, this compound did not induce retinal toxicity, a known side effect of chronic MER inhibition. Together, these data strongly suggest that AXL is a therapeutic target for overcoming drug resistance and that ER-851 is a promising candidate therapeutic agent for use against AXL-expressing antimitotic-resistant tumors.

Terconazole, an Azole Antifungal Drug, Increases Cytotoxicity in Antimitotic Drug-Treated Resistant Cancer Cells with Substrate-Specific P-gp Inhibitory Activity.

Azole antifungal drugs have been shown to enhance the cytotoxicity of antimitotic drugs in P-glycoprotein (P-gp)-overexpressing-resistant cancer cells. Herein, we examined two azole antifungal drugs, terconazole (TCZ) and butoconazole (BTZ), previously unexplored in resistant cancers. We found that both TCZ and BTZ increased cytotoxicity in vincristine (VIC)-treated P-gp-overexpressing drug-resistant KBV20C cancer cells. Following detailed analysis, low-dose VIC + TCZ exerted higher cytotoxicity than co-treatment with VIC + BTZ. Furthermore, we found that VIC + TCZ could increase apoptosis and induce G2 arrest. Additionally, low-dose TCZ could be combined with various antimitotic drugs to increase their cytotoxicity in P-gp-overexpressing antimitotic drug-resistant cancer cells. Moreover, TCZ exhibited P-gp inhibitory activity, suggesting that the inhibitory activity of P-gp plays a role in sensitization afforded by VIC + TCZ co-treatment. We also evaluated the cytotoxicity of 12 azole antifungal drugs at low doses in drug-resistant cancer cells. VIC + TCZ, VIC + itraconazole, and VIC + posaconazole exhibited the strongest cytotoxicity in P-gp-overexpressing KBV20C and MCF-7/ADR-resistant cancer cells. These drugs exerted robust P-gp inhibitory activity, accompanied by calcein-AM substrate efflux. Given that azole antifungal drugs have long been used in clinics, our results, which reposition azole antifungal drugs for treating P-gp-overexpressing-resistant cancer, could be employed to treat patients with drug-resistant cancer rapidly.

Counteractions of a Novel Hydroalcoholic Extract from Lens Culinaria against the Dexamethasone-Induced Osteoblast Loss of Native Murine Cells.

The cytoprotective effects of a novel hydroalcoholic extract (0.01-5 mg/mL) from Lens culinaria (Terre di Altamura Srl) were investigated within murine native skeletal muscle fibers, bone marrow cells, and osteoblasts, and in cell lines treated with the apoptotic agent staurosporine (2.14 × 10-6 M), the alkylating drug cisplatin (10-4 M), the topoisomerase I inhibitor irinotecan (10-4 M), the antimitotic pro-oxidant doxorubicin (10-6 M), and the immunosuppressant dexamethasone (2 × 10-6 M). An amount of 10g of plant material was used to obtain a 70% ethanol/water product, following two-step extraction, evaporation, lyophilization, and storage at -20 °C. For the murine osteoblasts, doxorubicin reduced survival by -65%, dexamethasone by -32% and -60% after 24 and 48 h of incubation time, respectively. The extract was effective in preventing the osteoblast count-reduction induced by dexamethasone; it was also effective at preventing the inhibition of mineralization induced by dexamethasone. Doxorubicin and cisplatin caused a significant reduction in cell growth by -77% for bone marrow cells, -43% for irinotecan, and -60% for dexamethasone, but there was no evidence for the cytoprotective effects of the extract in these cells. Staurosporine and doxorubicin caused a fiber death rate of >-40% after 18 and 24 h of incubation, yet the extract was not effective at preventing these effects. The extract was effective in preventing the staurosporine-induced reduction of HEK293 proliferation and colony formation in the crystal violet DNA staining and the clonogenic assays. It was also effective for the cisplatin-induced reduction in HEK293 cell proliferation. The extract, however, failed to protect the SHSY5Y neurons against cisplatin and irinotecan-induced cytotoxicity. A UV/VIS spectroscopy analysis showed three peaks at the wavelengths of 350, 260, and 190 nm, which correspond to flavonoids, proanthocyanins, salicylates, and AA, constituting the extract. These data suggest the possible development of this extract for use against dexamethasone-induced bone loss and renal chemotherapy-induced damage.

Evolution of Potential Antimitotic Stapled Peptides from Multiple Helical Peptide Stretches of the Tubulin Heterodimer Interface: Helix-Mimicking Stapled Peptide Tubulin Inhibitors.

Protein-protein interactions play a crucial role in microtubule dynamics. Microtubules are considered as a key target for the design and development of anticancer therapeutics, where inhibition of tubulin-tubulin interactions plays a crucial role. Here, we focused on a few key helical stretches at the interface of α,ß-tubulin heterodimers and developed a structural mimic of these helical peptides, which can serve as potent inhibitors of microtubule polymerization. To induce helicity, we have made stapled analogues of these sequences. Thereafter, we modified the lead sequences of the antimitotic stapled peptides with halo derivatives. It is observed that halo-substituted stapled peptides follow an interesting trend for the electronegativity of halogen atoms in interaction patterns with tubulin and a correlation in the toxicity profile. Remarkably, we found that para-fluorophenylalanine-modified stapled peptide is the most potent inhibitors, which perturbs microtubule dynamics, induces apoptotic death, and inhibits the growth of melanoma.

Early Pharmacological Profiling of Antiproliferative Compounds by Live Cell Imaging.

Natural products represent an excellent source of unprecedented anticancer compounds. However, the identification of the mechanism of action remains a major challenge. Several techniques and methodologies have been considered, but with limited success. In this work, we explored the combination of live cell imaging and machine learning techniques as a promising tool to depict in a fast and affordable test the mode of action of natural compounds with antiproliferative activity. To develop the model, we selected the non-small cell lung cancer cell line SW1573, which was exposed to the known antimitotic drugs paclitaxel, colchicine and vinblastine. The novelty of our methodology focuses on two main features with the highest relevance, (a) meaningful phenotypic metrics, and (b) fast Fourier transform (FFT) of the time series of the phenotypic parameters into their corresponding amplitudes and phases. The resulting algorithm was able to cluster the microtubule disruptors, and meanwhile showed a negative correlation between paclitaxel and the other treatments. The FFT approach was able to group the samples as efficiently as checking by eye. This methodology could easily scale to group a large amount of data without visual supervision.

Evaluation of the mechanism of action of albendazole on adult rat lungworm (Angiostrongyluscantonensis).

Albendazole is considered the anthelmintic of choice for the management of rat lungworm disease (neuroangiostrongyliasis), due to its broad spectrum of nematocidal activity and its ability to cross the blood-brain barrier. Albendazole binds to ß-tubulins, preventing their polymerization into microtubules, thereby corrupting the cascade of cell division at metaphase, which ultimately leads to the death of individual cells and eventually the death of the parasite. Inhibition of microtubule formation will also hinder the axoplasmic transport system, affecting the neuronal activities of the parasite. While this mechanism has been explicated in other parasitic and non-parasitic nematodes, it has never been evaluated in Angiostrongylus cantonensis. This study evaluates the antimitotic effects of albendazole sulphoxide (active metabolite) on the microtubules of adult A. cantonensis using the tubulin polymerization assay and measures its effects on worm viability using the colorimetric MTT assay. Three different concentrations of albendazole (62.5 µM, 250 µΜ, and 1 mM) were evaluated. We saw a statistically significant dose-dependent reduction in the band intensity of polymerized tubulins (or microtubules) (P = 0.019), suggesting that albendazole imparts its antimitotic effect in a dose-dependent manner. Similarly, our MTT assay showed a dose-dependent decrease in formazan intensity (proportional to cell viability), suggesting that the rate of nematocidal activity of albendazole is also proportional to its concentration. In compiling the results from both these experiments, a correlation between the microtubule assembly and worm viability is evident.

Low-Dose Rifabutin Increases Cytotoxicity in Antimitotic-Drug-Treated Resistant Cancer Cells by Exhibiting Strong P-gp-Inhibitory Activity.

The cytotoxicity of various antibiotics at low doses in drug-resistant cancer cells was evaluated. Low doses of rifabutin were found to markedly increase the cytotoxicity of various antimitotic drugs, such as vincristine (VIC), to P-glycoprotein (P-gp)-overexpressing antimitotic-drug-resistant KBV20C cells. Rifabutin was also found to exert high levels of P-gp-inhibitory activity at 4 and 24 h posttreatment, suggesting that the cytotoxicity of VIC + rifabutin was mainly due to the direct binding of rifabutin to P-gp and the reduction of VIC efflux by P-gp. The combination of VIC + rifabutin also increased early apoptosis, G2 arrest, and the DNA damaging marker, pH2AX protein. Interestingly, only the combination of VIC + rifabutin induced remarkable levels of cytotoxicity in resistant KBV20C cells, whereas other combinations (VIC + rifampin, VIC + rifapentine, and VIC + rifaximin) induced less cytotoxicity. Such finding suggests that rifabutin specifically increases the cytotoxicity of VIC in KBV20C cells, independent of the toxic effect of the ansamycin antibiotic. Only rifabutin had high P-gp-inhibitory activity, which suggests that its high P-gp-inhibitory activity led to the increased cytotoxicity of VIC + rifabutin. As rifabutin has long been used in the clinic, repositioning this drug for P-gp-overexpressing resistant cancer could increase the availability of treatments for patients with drug-resistant cancer.

Activation of STAT3 through combined SRC and EGFR signaling drives resistance to a mitotic kinesin inhibitor in glioblastoma.

Inhibitors of the mitotic kinesin Kif11 are anti-mitotics that, unlike vinca alkaloids or taxanes, do not disrupt microtubules and are not neurotoxic. However, development of resistance has limited their clinical utility. While resistance to Kif11 inhibitors in other cell types is due to mechanisms that prevent these drugs from disrupting mitosis, we find that in glioblastoma (GBM), resistance to the Kif11 inhibitor ispinesib works instead through suppression of apoptosis driven by activation of STAT3. This form of resistance requires dual phosphorylation of STAT3 residues Y705 and S727, mediated by SRC and epidermal growth factor receptor (EGFR), respectively. Simultaneously inhibiting SRC and EGFR reverses this resistance, and combined targeting of these two kinases in vivo with clinically available inhibitors is synergistic and significantly prolongs survival in ispinesib-treated GBM-bearing mice. We thus identify a translationally actionable approach to overcoming Kif11 inhibitor resistance that may work to block STAT3-driven resistance against other anti-cancer therapies as well.

Photopharmacology of Antimitotic Agents.

Antimitotic agents such as the clinically approved vinca alkaloids, taxanes and epothilone can arrest cell growth during interphase and are therefore among the most important drugs available for treating cancer. These agents suppress microtubule dynamics and thus interfere with intracellular transport, inhibit cell proliferation and promote cell death. Because these drugs target biological processes that are essential to all cells, they face an additional challenge when compared to most other drug classes. General toxicity can limit the applicable dose and therefore reduce therapeutic benefits. Photopharmacology aims to avoid these side-effects by introducing compounds that can be applied globally to cells in their inactive form, then be selectively induced to bioactivity in targeted cells or tissue during a defined time window. This review discusses photoswitchable analogues of antimitotic agents that have been developed by combining different photoswitchable motifs with microtubule-stabilizing or microtubule-destabilizing agents.

JAK2 Inhibitor, Fedratinib, Inhibits P-gp Activity and Co-Treatment Induces Cytotoxicity in Antimitotic Drug-Treated P-gp Overexpressing Resistant KBV20C Cancer Cells.

P-glycoprotein (P-gp) overexpression is one of the major mechanisms of multidrug resistance (MDR). Previously, co-treatment with Janus kinase 2 (JAK2) inhibitors sensitized P-gp-overexpressing drug-resistant cancer cells. In this study, we assessed the cytotoxic effects of JAK2 inhibitor, fedratinib, on drug-resistant KBV20C cancer cells. We found that co-treatment with fedratinib at low doses induced cytotoxicity in KBV20C cells treated with vincristine (VIC). However, fedratinib-induced cytotoxicity was little effect on VIC-treated sensitive KB parent cells, suggesting that these effects are specific to resistant cancer cells. Fluorescence-activated cell sorting (FACS), Western blotting, and annexin V analyses were used to further investigate fedratinib's mechanism of action in VIC-treated KBV20C cells. We found that fedratinib reduced cell viability, increased G2 arrest, and upregulated apoptosis when used as a co-treatment with VIC. G2 phase arrest and apoptosis in VIC-fedratinib-co-treated cells resulted from the upregulation of p21 and the DNA damaging marker pH2AX. Compared with dimethyl sulfoxide (DMSO)-treated cells, fedratinib-treated KBV20C cells showed two-fold higher P-gp-inhibitory activity, indicating that VIC-fedratinib sensitization is dependent on the activity of fedratinib. Similar to VIC, fedratinib co-treatment with other antimitotic drugs (i.e., eribulin, vinorelbine, and vinblastine) showed increased cytotoxicity in KBV20C cells. Furthermore, VIC-fedratinib had similar cytotoxic effects to co-treatment with other JAK2 inhibitors (i.e., VIC-CEP-33779 or VIC-NVP-BSK805) at the same dose; similar cytotoxic mechanisms (i.e., early apoptosis) were observed between treatments, suggesting that co-treatment with JAK2 inhibitors is generally cytotoxic to P-gp-overexpressing resistant cancer cells. Given that fedratinib is FDA-approved, our findings support its application in the co-treatment of P-gp-overexpressing cancer patients showing MDR.