Synergistic induction of mitotic pyroptosis and tumor remission by inhibiting proteasome and WEE family kinases.

Mitotic catastrophe (MC), which occurs under dysregulated mitosis, represents a fascinating tactic to specifically eradicate tumor cells. Whether pyroptosis can be a death form of MC remains unknown. Proteasome-mediated protein degradation is crucial for M-phase. Bortezomib (BTZ), which inhibits the 20S catalytic particle of proteasome, is approved to treat multiple myeloma and mantle cell lymphoma, but not solid tumors due to primary resistance. To date, whether and how proteasome inhibitor affected the fates of cells in M-phase remains unexplored. Here, we show that BTZ treatment, or silencing of PSMC5, a subunit of 19S regulatory particle of proteasome, causes G2- and M-phase arrest, multi-polar spindle formation, and consequent caspase-3/GSDME-mediated pyroptosis in M-phase (designated as mitotic pyroptosis). Further investigations reveal that inhibitor of WEE1/PKMYT1 (PD0166285), but not inhibitor of ATR, CHK1 or CHK2, abrogates the BTZ-induced G2-phase arrest, thus exacerbates the BTZ-induced mitotic arrest and pyroptosis. Combined BTZ and PD0166285 treatment (named BP-Combo) selectively kills various types of solid tumor cells, and significantly lessens the IC50 of both BTZ and PD0166285 compared to BTZ or PD0166285 monotreatment. Studies using various mouse models show that BP-Combo has much stronger inhibition on tumor growth and metastasis than BTZ or PD0166285 monotreatment, and no obvious toxicity is observed in BP-Combo-treated mice. These findings disclose the effect of proteasome inhibitors in inducing pyroptosis in M-phase, characterize pyroptosis as a new death form of mitotic catastrophe, and identify dual inhibition of proteasome and WEE family kinases as a promising anti-cancer strategy to selectively kill solid tumor cells.

Elevating PLK1 overcomes BETi resistance in prostate cancer via triggering BRD4 phosphorylation-dependent degradation in mitosis.

Bromodomain-containing protein 4 (BRD4) has emerged as a promising therapeutic target in prostate cancer (PCa). Understanding the mechanisms of BRD4 stability could enhance the clinical response to BRD4-targeted therapy. In this study, we report that BRD4 protein levels are significantly decreased during mitosis in a PLK1-dependent manner. Mechanistically, we show that BRD4 is primarily phosphorylated at T1186 by the CDK1/cyclin B complex, recruiting PLK1 to phosphorylate BRD4 at S24/S1100, which are recognized by the APC/CCdh1 complex for proteasome pathway degradation. We find that PLK1 overexpression lowers SPOP mutation-stabilized BRD4, consequently rendering PCa cells re-sensitized to BRD4 inhibitors. Intriguingly, we report that sequential treatment of docetaxel and JQ1 resulted in significant inhibition of PCa. Collectively, the results support that PLK1-phosphorylated BRD4 triggers its degradation at M phase. Sequential treatment of docetaxel and JQ1 overcomes BRD4 accumulation-associated bromodomain and extra-terminal inhibitor (BETi) resistance, which may shed light on the development of strategies to treat PCa.

Caspase-mediated AURKA cleavage at Asp132 is essential for paclitaxel to elicit cell apoptosis.

Background: Aurora kinase A (AURKA) is a potent oncogene that is often aberrantly expressed during tumorigenesis, and is associated with chemo-resistance in various malignancies. However, the role of AURKA in chemo-resistance remains largely elusive. Methods: The cleavage of AURKA upon viral infection or apoptosis stimuli was assesed by immunoblotting assays in several cancer cells or caspase deficient cell line models. The effect of AURKA cleavage at Asp132 on mitosis was explored by live cell imaging and immunofluorescence staining experiments. The role of Asp132-cleavage of AURKA induced by the chemotherapy drug paclitaxel was investigated using TUNEL, immunohistochemistry assay in mouse tumor xenograft model and patient tissues. Results: The proteolytic cleavage of AURKA at Asp132 commonly occurs in several cancer cell types, regardless of viral infection or apoptosis stimuli. Mechanistically, caspase 3/7/8 cleave AURKA at Asp132, and the Asp132-cleaved forms of AURKA promote cell apoptosis by disrupting centrosome formation and bipolar spindle assembly in metaphase during mitosis. The AURKAD132A mutation blocks the expression of cleaved caspase 3 and EGR1, which leads to reduced therapeutic effects of paclitaxel on colony formation and malignant growth of tumor cells in vitro and in vivo using a murine xenograft model and cancer patients. Conclusions: This study reveals that caspase-mediated AURKAD132 proteolysis is essential for paclitaxel to elicit cell apoptosis and indicates that AURKAD132 is a potential key target for chemotherapy.

Toto-Cell: A new software to analyze cellular events during video-microscopy.

Video-microscopy is a technology widely used to follow, in a single cell manner, cell behavior. A number of new studies are searching a way to track these behaviors by artificial intelligence; unfortunately some real-time events still have to be track manually. For that reason, we developed a software that helps the experimenter to analyze collected data. Toto-cell is very simple to use and it can be adapted at different type of analyses or treatments. It allows a wide new range of parameters that were nearly impossible to calculate only by hand. We thus developed this new software using HEC-1-A endometrial cell line to track different cellular parameters such as: the number of normal/abnormal mitosis, the ratio per day of death, mitosis, cell fusions or finally the length between two mitosis cycles. We treated our cells with cisplatin, doxorubicin or AZD5363 (an Akt inhibitor) to obtain different cellular events. What emerged is a huge heterogeneity for these analyzed parameters between the cells in a single treatment which is clearly demonstrated by the results provided by Toto-Cell. In conclusion, our software is an important tool to facilitate the analysis of video-microscopy, in a quantifying and qualifying manner. It enables a higher accuracy when compared to manual calculations.

Suppressing Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 Activity to Enhance the Effectiveness of Anti-Cancer Drugs That Induce Multipolar Mitotic Spindles.

Paclitaxel induces multipolar spindles at clinically relevant doses but does not substantially increase mitotic indices. Paclitaxel's anti-cancer effects are hypothesized to occur by promoting chromosome mis-segregation on multipolar spindles leading to apoptosis, necrosis and cyclic-GMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) pathway activation in daughter cells, leading to secretion of type I interferon (IFN) and immunogenic cell death. Eribulin and vinorelbine have also been reported to cause increases in multipolar spindles in cancer cells. Recently, suppression of Anaphase-Promoting Complex/Cyclosome-Cell Division Cycle 20 (APC/C-CDC20) activity using CRISPR/Cas9 mutagenesis has been reported to increase sensitivity to Kinesin Family 18a (KIF18a) inhibition, which functions to suppress multipolar mitotic spindles in cancer cells. We propose that a way to enhance the effectiveness of anti-cancer agents that increase multipolar spindles is by suppressing the APC/C-CDC20 to delay, but not block, anaphase entry. Delaying anaphase entry in genomically unstable cells may enhance multipolar spindle-induced cell death. In genomically stable healthy human cells, delayed anaphase entry may suppress the level of multipolar spindles induced by anti-cancer drugs and lower mitotic cytotoxicity. We outline specific combinations of molecules to investigate that may achieve the goal of enhancing the effectiveness of anti-cancer agents.

Targeting mTOR and survivin concurrently potentiates radiation therapy in renal cell carcinoma by suppressing DNA damage repair and amplifying mitotic catastrophe.

BACKGROUND: Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. EXPERIMENTAL DESIGN: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. RESULTS: EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. CONCLUSION: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Anlotinib synergizes with venetoclax to induce mitotic catastrophe in acute myeloid leukemia.

Venetoclax is a BCL2-targeted drug employed in treating various cancers, particularly hematologic malignancies. Venetoclax combination therapies are increasingly recognized as promising treatment strategies for acute myeloid leukemia (AML). In this study, we conducted an unbiased drug screen and identified anlotinib, a promising multi-targeted receptor tyrosine kinase inhibitor with oral activity currently utilized in the treatment of solid tumor, as a potent enhancer of venetoclax's anticancer activity in AML. Our investigation encompassed AML cell lines, primary cells, and mouse models, demonstrating effective low-dose combination therapy of anlotinib and venetoclax with minimal cytopenia or organ damage. Proteomic analysis revealed abnormal mitotic signals induced by this combination in AML cells. Mechanistically, anlotinib synergized with venetoclax by suppressing ARPP19 protein, leading to sustained activation of PP2A-B55δ. This inhibited AML cells from entering the mitotic phase, culminating in mitotic catastrophe and apoptosis. Additionally, we identified a specific synthetic lethal vulnerability in AML involving an ARPP19 mutation at S62 phosphorylation. These findings underscore the therapeutic potential of anlotinib and venetoclax combination therapy in AML, warranting further clinical investigation.

Approaches to selective and potent inhibition of glioblastoma by vanadyl complexes: Inducing mitotic catastrophe and methuosis.

Drug resistance has been a major problem for cancer chemotherapy, especially for glioblastoma multiforme that is aggressive, heterogeneous and recurrent with <3% of a five-year survival and limited methods of clinical treatment. To overcome the problem, great efforts have recently been put in searching for agents inducing death of tumor cells via various non-apoptotic pathways. In the present work, we report for the first time that vanadyl complexes, i.e. bis(acetylacetonato)oxidovanadium (IV) (VO(acac)2), can cause mitotic catastrophe and methuotic death featured by catastrophic macropinocytic vacuole accumulation particularly in glioblastoma cells (GCs). Hence, VO(acac)2 strongly suppressed growth of GCs with both in vitro (IC50 = 4-6 µM) and in vivo models, and is much more potent than the current standard-of-care drug Temozolomide. The selective index is as high as ∼10 or more on GCs over normal neural cells. Importantly, GCs respond well to vanadium treatment regardless whether they are carrying IDH1 wild type gene that causes drug resistance. VO(acac)2 may induce methuosis via the Rac-Mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase (JNK) signaling pathway. Furthermore, VO(acac)2-induced methuosis is not through a immunogenicity mechanism, making vanadyl complexes safe for interventional therapy. Overall, our results may encourage development of novel vanadium complexes promising for treatment of neural malignant tumor cells.

Signatures of epigenetic, biological and mitotic age acceleration and telomere shortening are associated with arsenic-induced skin lesions.

Chronic arsenic exposure is a global health hazard significantly associated with the development of deleterious cutaneous changes and increased keratinocyte cancer risk. Although arsenic exposure is associated with broad-scale cellular and molecular changes, gaps exist in understanding how these changes impact the skin and facilitate malignant transformation. Recently developed epigenetic "clocks" can accurately predict chronological, biological and mitotic age, as well as telomere length, on the basis of tissue DNA methylation state. Deviations of predicted from expected age (epigenetic age dysregulation) have been associated with numerous complex diseases, increased all-cause mortality and higher cancer risk. We investigated the ability of these algorithms to detect molecular changes associated with chronic arsenic exposure in the context of associated skin lesions. To accomplish this, we utilized a multi-algorithmic approach incorporating seven "clocks" (Horvath, Skin&Blood, PhenoAge, PCPhenoAge, GrimAge, DNAmTL and epiTOC2) to analyze peripheral blood of pediatric and adult cohorts of arsenic-exposed (n = 84) and arsenic-naïve (n = 33) individuals, among whom n = 18 were affected by skin lesions. Arsenic-exposed adults with skin lesions exhibited accelerated epigenetic (Skin&Blood: + 7.0 years [95% CI 3.7; 10.2], q = 6.8 × 10-4), biological (PhenoAge: + 5.8 years [95% CI 0.7; 11.0], q = 7.4 × 10-2, p = 2.8 × 10-2) and mitotic age (epiTOC2: + 19.7 annual cell divisions [95% CI 1.8; 37.7], q = 7.4 × 10-2, p = 3.2 × 10-2) compared to healthy arsenic-naïve individuals; and accelerated epigenetic age (Skin&Blood: + 2.8 years [95% CI 0.2; 5.3], q = 2.4 × 10-1, p = 3.4 × 10-2) compared to lesion-free arsenic-exposed individuals. Moreover, lesion-free exposed adults exhibited accelerated Skin&Blood age (+ 4.2 [95% CI 1.3; 7.1], q = 3.8 × 10-2) compared to their arsenic-naïve counterparts. Compared to the pediatric group, arsenic-exposed adults exhibited accelerated epigenetic (+ 3.1 to 4.4 years (95% CI 1.2; 6.4], q = 2.4 × 10-4-3.1 × 10-3), biological (+ 7.4 to 7.8 years [95% CI 3.0; 12.1] q = 1.6 × 10-3-2.8 × 10-3) and mitotic age (+ 50.0 annual cell divisions [95% CI 15.6; 84.5], q = 7.8 × 10-3), as well as shortened telomere length (- 0.23 kilobases [95% CI - 0.13; - 0.33], q = 2.4 × 10-4), across all seven algorithms. We demonstrate that lifetime arsenic exposure and presence of arsenic-associated skin lesions are associated with accelerated epigenetic, biological and mitotic age, and shortened telomere length, reflecting altered immune signaling and genomic regulation. Our findings highlight the usefulness of DNA methylation-based algorithms in identifying deleterious molecular changes associated with chronic exposure to the heavy metal, serving as potential prognosticators of arsenic-induced cutaneous malignancy.

Inhibition of the RPS6KA1/FoxO1 signaling axis by hydroxycitric acid attenuates HFD-induced obesity through MCE suppression.

BACKGROUND: Because obesity is associated with a hyperplasia-mediated increase in adipose tissue, inhibiting cell proliferation during mitotic clonal expansion (MCE) is a leading strategy for preventing obesity. Although (-)-hydroxycitric acid (HCA) is used to control obesity, the molecular mechanisms underlying its effects on MCE are poorly understood. PURPOSE: This study aimed to investigate the potential effects of HCA on MCE and underlying molecular mechanisms affecting adipogenesis and obesity improvements. METHODS: Preadipocyte cell line, 3T3-L1, were treated with HCA; oil red O, cell proliferation, cell cycle, and related alterations in signaling pathways were examined. High-fat diet (HFD)-fed mice were administered HCA for 12 weeks; body and adipose tissues weights were evaluated, and the regulation of signaling pathways in epidydimal white adipose tissue were examined in vivo. RESULTS: Here, we report that during MCE, HCA attenuates the proliferation of the preadipocyte cell line, 3T3-L1, by arresting the cell cycle at the G0/G1 phase. In addition, HCA markedly inhibits Forkhead Box O1 (FoxO1) phosphorylation, thereby inducing the expression of cyclin-dependent kinase inhibitor 1B and suppressing the levels of cyclin-dependent kinase 2, cyclin E1, proliferating cell nuclear antigen, and phosphorylated retinoblastoma. Importantly, we found that ribosomal protein S6 kinase A1 (RPS6KA1) influences HCA-mediated inactivation of FoxO1 and its nuclear exclusion. An animal model of obesity revealed that HCA reduced high-fat diet-induced obesity by suppressing adipocyte numbers as well as epididymal and mesenteric white adipose tissue mass, which is attributed to the regulation of RPS6KA1, FoxO1, CDKN1B and PCNA that had been consistently identified in vitro. CONCLUSIONS: These findings provide novel insights into the mechanism by which HCA regulates adipogenesis and highlight the RPS6KA1/FoxO1 signaling axis as a therapeutic target for obesity.

Simvastatin activates the spindle assembly checkpoint and causes abnormal cell division by modifying small GTPases.

Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which is a rate-limiting enzyme of the cholesterol synthesis pathway. It has been used clinically as a lipid-lowering agent to reduce low-density lipoprotein (LDL) cholesterol levels. In addition, antitumor activity has been demonstrated. Although simvastatin attenuates the prenylation of small GTPases, its effects on cell division in which small GTPases play an important role, have not been examined as a mechanism underlying its cytostatic effects. In this study, we determined its effect on cell division. Cell cycle synchronization experiments revealed a delay in mitotic progression in simvastatin-treated cells at concentrations lower than the IC50. Time-lapse imaging analysis indicated that the duration of mitosis, especially from mitotic entry to anaphase onset, was prolonged. In addition, simvastatin increased the number of cells exhibiting misoriented anaphase/telophase and bleb formation. Inhibition of the spindle assembly checkpoint (SAC) kinase Mps1 canceled the mitotic delay. Additionally, the number of cells exhibiting kinetochore localization of BubR1, an essential component of SAC, was increased, suggesting an involvement of SAC in the mitotic delay. Enhancement of F-actin formation and cell rounding at mitotic entry indicates that cortical actin dynamics were affected by simvastatin. The cholesterol removal agent methyl-ß-cyclodextrin (MßCD) accelerated mitotic progression differently from simvastatin, suggesting that cholesterol loss from the plasma membrane is not involved in the mitotic delay. Of note, the small GTPase RhoA, which is a critical factor for cortical actin dynamics, exhibited upregulated expression. In addition, Rap1 was likely not geranylgeranylated. Our results demonstrate that simvastatin affects actin dynamics by modifying small GTPases, thereby activating the spindle assembly checkpoint and causing abnormal cell division.

Multiplexed single-cell lineage tracing of mitotic kinesin inhibitor resistance in glioblastoma.

Glioblastoma (GBM) is a deadly brain tumor, and the kinesin motor KIF11 is an attractive therapeutic target with roles in proliferation and invasion. Resistance to KIF11 inhibitors, which has mainly been studied in animal models, presents significant challenges. We use lineage-tracing barcodes and single-cell RNA sequencing to analyze resistance in patient-derived GBM neurospheres treated with ispinesib, a potent KIF11 inhibitor. Similar to GBM progression in patients, untreated cells lose their neural lineage identity and become mesenchymal, which is associated with poor prognosis. Conversely, cells subjected to long-term ispinesib treatment exhibit a proneural phenotype. We generate patient-derived xenografts and show that ispinesib-resistant cells form less aggressive tumors in vivo, even in the absence of drug. Moreover, treatment of human ex vivo GBM slices with ispinesib demonstrates phenotypic alignment with in vitro responses, underscoring the clinical relevance of our findings. Finally, using retrospective lineage tracing, we identify drugs that are synergistic with ispinesib.

Antitumoral Activity of the Universal Methyl Donor S-Adenosylmethionine in Glioblastoma Cells.

Glioblastoma (GBM), the most frequent and lethal brain cancer in adults, is characterized by short survival times and high mortality rates. Due to the resistance of GBM cells to conventional therapeutic treatments, scientific interest is focusing on the search for alternative and efficient adjuvant treatments. S-Adenosylmethionine (AdoMet), the well-studied physiological methyl donor, has emerged as a promising anticancer compound and a modulator of multiple cancer-related signaling pathways. We report here for the first time that AdoMet selectively inhibited the viability and proliferation of U87MG, U343MG, and U251MG GBM cells. In these cell lines, AdoMet induced S and G2/M cell cycle arrest and apoptosis and downregulated the expression and activation of proteins involved in homologous recombination DNA repair, including RAD51, BRCA1, and Chk1. Furthermore, AdoMet was able to maintain DNA in a damaged state, as indicated by the increased γH2AX/H2AX ratio. AdoMet promoted mitotic catastrophe through inhibiting Aurora B kinase expression, phosphorylation, and localization causing GBM cells to undergo mitotic catastrophe-induced death. Finally, AdoMet inhibited DNA repair and induced cell cycle arrest, apoptosis, and mitotic catastrophe in patient-derived GBM cells. In light of these results, AdoMet could be considered a potential adjuvant in GBM therapy.

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.

The Application of Kinesin Inhibitors in Medical Issues.

Kinesins are a group of motor proteins in charge of several crucial functions in the cell. These proteins often bind to microtubules and perform their functions using the energy produced by ATP hydrolysis. One function of mitotic kinesin, a subclass of kinesin that is expressed during cell division at the mitotic phase, is to create the mitotic spindle. Uncontrolled cell growth is one trait of cancerous cells. Traditional anticancer medications still used in clinics include taxanes (paclitaxel) and vinca alkaloids (vincristine, vinblastine), which interfere with microtubule dynamics. However, because non-dividing cells like post-mitotic neurons contain microtubules, unwanted side effects like peripheral neuropathy are frequently found in patients taking these medications. More than ten members of the mitotic kinesin family play distinct or complementary roles during mitosis. The mitotic kinesin family's KSP, or Eg5, is regarded as its most dramatic target protein. The current work systematically reviews the use of kinesin inhibitors in the medical field. The challenges of KSP and the practical solutions are also examined, and the outcomes of the previous works are reported. The significant gaps and shortcomings of the related works are also highlighted, which can be an onset topic for future works.

Src inhibition induces mitotic arrest associated with chromosomal passenger complex.

The non-receptor tyrosine kinase Src plays a key role in cell division, migration, adhesion, and survival. Src is overactivated in several cancers, where it transmits signals that promote cell survival, mitosis, and other important cancer hallmarks. Src is therefore a promising target in cancer therapy, but the underlying mechanisms are still uncertain. Here we show that Src is highly conserved across different species. Src expression increases during mitosis and is localized to the chromosomal passenger complex. Knockdown or inhibition of Src induces multipolar spindle formation, resulting in abnormal expression of the Aurora B and INCENP components of the chromosomal passenger complex. Molecular mechanism studies have found that Src interacts with and phosphorylates INCENP. This then leads to incorrect chromosome arrangement and segregation, resulting in cell division failure. Herein, Src and chromosomal passenger complex co-localize and Src inhibition impedes mitotic progression by inducing multipolar spindle formation. These findings provide novel insights into the molecular basis for using Src inhibitors to treat cancer.

Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways.

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

The phytochemical, corynoline, diminishes Aurora kinase B activity to induce mitotic defect and polyploidy.

Plants are a rich source for bioactive compounds. However, plant extracts can harbor a mixture of bioactive molecules that promote divergent phenotypes and potentially have confounding effects in bioassays. Even with further purification and identification, target deconvolution can be challenging. Corynoline and acetylcorynoline, are phytochemicals that were previously isolated through a screen for compounds able to induce mitotic arrest and polyploidy in oncogene expressing retinal pigment epithelial (RPE) cells. Here, we shed light on the mechanism by which these phytochemicals can attack human cancer cells. Mitotic arrest was coincident to the induction of centrosome amplification and declustering, causing multi-polar spindle formation. Corynoline was demonstrated to have true centrosome declustering activity in a model where A549 cells were chemically induced to have more than a regular complement of centrosomes. Corynoline could inhibit the centrosome clustering required for pseudo-bipolar spindle formation in these cells. The activity of AURKB, but not AURKA or polo-like kinase 4, was diminished by corynoline. It only partially inhibited AURKB, so it may be a partial antagonist or corynoline may work upstream on an unknown regulator of AURKB activity or localization. Nonetheless, corynoline and acetylcorynoline inhibited the viability of a variety of human cancer derived cell lines. These phytochemicals could serve as prototypes for a next-generation analog with improved potency, selectivity or in vivo bioavailability. Such an analog could be useful as a non-toxic component of combination therapies where inhibiting the chromosomal passenger protein complex is desired.

Rhenium Perrhenate (188ReO4) Induced Apoptosis and Reduced Cancerous Phenotype in Liver Cancer Cells.

Recurrence in hepatocellular carcinoma (HCC) after conventional treatments is a crucial challenge. Despite the promising progress in advanced targeted therapies, HCC is the fourth leading cause of cancer death worldwide. Radionuclide therapy can potentially be a practical targeted approach to address this concern. Rhenium-188 (188Re) is a ß-emitting radionuclide used in the clinic to induce apoptosis and inhibit cell proliferation. Although adherent cell cultures are efficient and reliable, appropriate cell-cell and cell-extracellular matrix (ECM) contact is still lacking. Thus, we herein aimed to assess 188Re as a potential therapeutic component for HCC in 2D and 3D models. The death rate in treated Huh7 and HepG2 lines was significantly higher than in untreated control groups using viability assay. After treatment with 188ReO4, Annexin/PI data indicated considerable apoptosis induction in HepG2 cells after 48 h but not Huh7 cells. Quantitative RT-PCR and western blotting data also showed increased apoptosis in response to 188ReO4 treatment. In Huh7 cells, exposure to an effective dose of 188ReO4 led to cell cycle arrest in the G2 phase. Moreover, colony formation assay confirmed post-exposure growth suppression in Huh7 and HepG2 cells. Then, the immunostaining displayed proliferation inhibition in the 188ReO4-treated cells on 3D scaffolds of liver ECM. The PI3-AKT signaling pathway was activated in 3D culture but not in 2D culture. In nude mice, Huh7 cells treated with an effective dose of 188ReO4 lost their tumor formation ability compared to the control group. These findings suggest that 188ReO4 can be a potential new therapeutic agent against HCC through induction of apoptosis and cell cycle arrest and inhibition of tumor formation. This approach can be effectively combined with antibodies and peptides for more selective and personalized therapy.

In Vitro Anticancer Screening and Preliminary Mechanistic Study of A-Ring Substituted Anthraquinone Derivatives.

Anthraquinone derivatives exhibit various biological activities, e.g., antifungal, antibacterial and in vitro antiviral activities. They are naturally produced in many fungal and plant families such as Rhamnaceae or Fabaceae. Furthermore, they were found to have anticancer activity, exemplified by mitoxantrone and pixantrone, and many are well known redox-active compounds. In this study, various nature inspired synthetic anthraquinone derivatives were tested against colon, prostate, liver and cervical cancer cell lines. Most of the compounds exhibit anticancer effects against all cell lines, therefore the compounds were further studied to determine their IC50-values. Of these compounds, 1,4-bis(benzyloxy)-2,3-bis(hydroxymethyl)anthracene-9,10-dione (4) exhibited the highest cytotoxicity against PC3 cells and was chosen for a deeper look into its mechanism of action. Based on flow cytometry, the compound was proven to induce apoptosis through the activation of caspases and to demolish the ROS/RNS and NO equilibrium in the PC3 cell line. It trapped cells in the G2/M phase. Western blotting was performed for several proteins related to the effects observed. Compound 4 enhanced the production of PARP and caspase-3. Moreover, it activated the conversion of LC3A/B-I to LC3A/B-II showing that also autophagy plays a role in its mechanism of action, and it caused the phosphorylation of p70 s6 kinase.