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Effect of 1 on α-tubulin and β-tubulin expresion. H630 A. and H630R1 B. cells were seeded on glass coverslips, incubated with 1 (20 nM) for 18 h, then fixed and processed for confocal microscopy as described in the Materials and Methods section.
Source publication
The novel, chemically stabilized disorazole analog, (-)-CP2-disorazole C1 (1) displayed potent anti-proliferative activity against a broad-spectrum of human colorectal cancer cells. HCT15 and H630R1 cell lines expressing high basal levels of the ABCB1 protein, known to cause multi-drug resistance, were also sensitive to growth inhibition by 1 but w...
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... next investigated the effect of 1 on tubulin organization in intact cells and the microtubule structure of cells was visualized via immunocytochemistry. As shown in Figure 3, both α-tubulin and β-tubulin microtubules formed an intact network of tubules extending into the lamellipodium in untreated cells. Treatment of H630 and H630R1 cells with 1 resulted in significant disruption of both tubulin subtypes, loss of cellular structure, and formation of cell membrane rounding. Following drug treatment, both α-tubulin and β-tubulin appeared as a punctate dot-staining ...
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Citations
... G 2 /M arrest is a mechanism widely exploited by the pharmaceutical industry to prevent cell division. The literature shows its application and efficiency in 3D model of prostate cancer (axitinib) [69], lung cancer (cabazitaxel) [70], colorectal cancer (docetaxel) [71], and breast cancer (epirubicin and hyaluronidase) [72,73]. A similar effect was observed in treatments with rucaparib in PC3 and LNCaP (prostate cancer) cell lines, delaying spheroid growth by increasing the rate of cell death [74]. ...
The monolayer (two-dimensional or 2D) cell culture, while widely used, lacks fidelity
in replicating vital cell interactions seen in vivo, leading to a shift toward three-dimensional (3D) models. Although monolayers offer simplicity and cost-effectiveness, spheroids mimic cellular environments better. This is due to its nutrient gradients, which influence drug penetration and provide a more accurate reflection of clinical scenarios than monolayers. Consequently, 3D models
are crucial in drug development, especially for anti-cancer therapeutics, enabling the screening of cell cycle inhibitors and combination therapies vital for
heterogeneous tumor populations. Inhibiting processes like migration and invasion often require drugs targeting the cytoskeleton, which can exhibit dual functionality with cell cycle inhibitors. Therapeutic approaches with promising anticancer
potential often exhibit reduced efficacy in 3D cell culture compared to their performance in monolayer settings, primarily due to the heightened complexity inherent in this system. In the face of this scenario, this review aims to survey existing knowledge on compounds utilized in both 2D and 3D cell cultures, assessing their responses across different culture types and discerning the
implications for drug screening, particularly those impacting the cell cycle and cytoskeletal dynamics.
... Generally, caspase-8 and -9 are activated during extrinsic and intrinsic apoptotic pathway, respectively, and the extrinsic pathway can converge with intrinsic pathway through the truncation of Bid, a key player in the crosstalk between intrinsic and extrinsic pathways [1,17,19]. In addition, necrosis and cell cycle arrest have been characterized as important cellular processes responsible for the action of many chemotherapeutic drugs [20][21][22][23]. All these cellular processes are closely correlated with the activity of various chemotherapeutic reagents, including arsenic compounds, of inducing DNA damage, which in turn leads to the manipulation of transcription factors such as p53 and c-Myc as well as their respective downstream molecules, and ultimately contributes to cell death and/or cell cycle arrest [24,25]. ...
... By contrast, only a small increase in the expression of FEN1, a factor in DNA damage repair [35,37], was confirmed, suggesting the compensatory increase in response to the DNA damage induced by darinaparsin. DNA damage can halt cell cycle progression by upregulating p53 and/or downregulating c-Myc and can subsequently result in G2/M arrest as well as apoptosis [20][21][22][23]38]. The abnormal expression of cdc25C has been linked to disease progression and poor prognosis in many cancer types, and its downregulation could induce cell cycle arrest in the G2/M phase in response to DNA damage via p53mediated signal transduction [45]. ...
... In addition, downregulation of c-Myc has been shown to modulate the Rb/E2F pathway, leading to G2/M arrest [21]. It has been shown that non-phosphorylated Rb, the active form of Rb, can bind E2F1 and consequently result in the inactivation of E2F1 and inhibition of the cell cycle [21,23,47]. In the current study, the activation of p53 and downregulation of c-Myc were first observed in NB4 cells treated by darinaparsin. ...
To explore the molecular mechanisms of action underlying the antileukemia activities of darinaparsin, an organic arsenical approved for the treatment of peripheral T–cell lymphoma in Japan, cytotoxicity of darinaparsin was evaluated in leukemia cell lines NB4, U-937, MOLT-4 and HL-60. Darinaparsin was a more potent cytotoxic than sodium arsenite, and induced apoptosis/necrosis in NB4 and HL-60 cells. In NB4 cells exhibiting the highest susceptibility to darinaparsin, apoptosis induction was accompanied by the activation of caspase-8/-9/-3, a substantial decrease in Bid expression, and was suppressed by Boc-D-FMK, a pancaspase inhibitor, suggesting that darinaparsin triggered a convergence of the extrinsic and intrinsic pathways of apoptosis via Bid truncation. A dramatic increase in the expression level of γH2AX, a DNA damage marker, occurred in parallel with G2/M arrest. Activation of p53 and the inhibition of cdc25C/cyclin B1/cdc2 were concomitantly observed in treated cells. Downregulation of c-Myc, along with inactivation of E2F1 associated with the activation of Rb, was observed, suggesting the critical roles of p53 and c-Myc in darinaparsin-mediated G2/M arrest. Trolox, an antioxidative reagent, suppressed the apoptosis induction but failed to correct G2/M arrest, suggesting that oxidative stress primarily contributed to apoptosis induction. Suppression of Notch1 signaling was also confirmed. Our findings provide novel insights into molecular mechanisms underlying the cytotoxicity of darinaparsin and strong rationale for its new clinical application for patients with different types of cancer.
... Their formation apparently occurs due to disturbances in lipid metabolism and is facilitated by oxidative stress, mediated by reactive oxygen species, as evident from the inhibition of thorn formation following treatment with antioxidants. The thorns morphologically resemble neurite outgrowth in sensory ganglia exposed to nerve growth factor [19] and endothelial sprouting [20], but are morphologically and mechanistically distinct. Thorns exhibit blue autofluorescence, which may offer insight into their composition. ...
The in vivo-relevant phenotype of 3D liver spheroids allows for long-term studies of, e.g., novel mechanisms of chronic drug-induced liver toxicity. Using this system, we present a novel drug-induced stress response in human and murine hepatocyte spheroids, wherein long slender filaments form after chronic treatment with four different drugs, of which three are PPARα antagonists. The morphology of the thorns varies between donors and the compounds used. They are mainly composed of diverse protein fibres, which are glycosylated. Their formation is inhibited by treatment with fatty acids or antioxidants. Treatment of mice with GW6471 revealed changes in gene and protein expression, such as those in the spheroids. In addition, similar changes in keratin expression were seen following the treatment of hepatotoxic drugs, including aflatoxin B1, paracetamol, chlorpromazine, cyclosporine, and ketoconazole. We suggest that thorn formation may be indicative of hepatocyte metaplasia in response to toxicity and that more focus should be placed on alterations of ECM-derived protein expression as biomarkers of liver disease and chronic drug-induced hepatotoxicity, changes that can be studied in stable in vivo-like hepatic cell systems, such as the spheroids.
... Preclinical immunoblot experiments of α-and β-tubulin expression in human RKO colon cancer cells and the tubulin polymerization assay were performed as previously described [12,18]. With respect to the trial-related assessments, tissue samples (plasma, PBMCs, and skin Biopsies were obtained at C1D-15, C1D1, and C1D15, formalin-fixed, paraffin-embedded and slides were stained with indicated antibodies. ...
Purpose
We investigated the combination of tivantinib, a c-MET tyrosine kinase inhibitor (TKI), and bevacizumab, an anti-VEGF-A antibody.
Methods
Patients with advanced solid tumors received bevacizumab (10 mg/kg intravenously every 2 weeks) and escalating doses of tivantinib (120–360 mg orally twice daily). In addition to safety and preliminary efficacy, we evaluated pharmacokinetics of tivantinib and its metabolites, as well as pharmacodynamic biomarkers in peripheral blood and skin.
Results
Eleven patients received the combination treatment, which was generally well tolerated. The main dose-limiting toxicity was grade 3 hypertension, which was observed in four patients. Other toxicities included lymphopenia and electrolyte disturbances. No exposure-toxicity relationship was observed for tivantinib or metabolites. No clinical responses were observed. Mean levels of the serum cytokine bFGF increased (p = 0.008) after the bevacizumab-only lead-in and decreased back to baseline (p = 0.047) after addition of tivantinib. Tivantinib reduced levels of both phospho-MET (7/11 patients) and tubulin (4/11 patients) in skin.
Conclusions
The combination of tivantinib and bevacizumab produced toxicities that were largely consistent with the safety profiles of the individual drugs. The study was terminated prior to establishment of the recommended phase II dose (RP2D) due to concerns regarding the mechanism of tivantinib, as well as lack of clinical efficacy seen in this and other studies. Tivantinib reversed the upregulation of bFGF caused by bevacizumab, which has been considered a potential mechanism of resistance to therapies targeting the VEGF pathway. The findings from this study suggest that the mechanism of action of tivantinib in humans may involve inhibition of both c-MET and tubulin expression.
Trial registration
NCT01749384 (First posted 12/13/2012).
... The mechanisms of resistance to this class of compounds include overexpression of the drug efflux pump protein ABCB1, microtubule cytoskeletal changes, and over expression of specific β-tubulin isotype and microtubule-associated proteins. The microtubulestabilizing agents, such as epothilones, have demonstrated similar activity in ABCB1-overexpressing cells [18]. But ABCB1 was not selected as strong features by ISIS and ENR. ...
Background:
Prediction of drug response based on multi-omics data is a crucial task in the research of personalized cancer therapy.
Results:
We proposed an iterative sure independent ranking and screening (ISIRS) scheme to select drug response-associated features and applied it to the Cancer Cell Line Encyclopedia (CCLE) dataset. For each drug in CCLE, we incorporated multi-omics data including copy number alterations, mutation and gene expression and selected up to 50 features using ISIRS. Then a linear regression model based on the selected features was exploited to predict the drug response. Cross validation test shows that our prediction accuracies are higher than existing methods for most drugs.
Conclusions:
Our study indicates that the features selected by the marginal utility measure, which measures the conditional probability of drug responses given the feature, are helpful for drug response prediction.
... In addition, animal models of spinal cord injury have indicated that ES influences the proliferation and directional migration of nerve cells via the Wnt/β-catenin signaling pathway (18)(19)(20). In the field of oncology, it has been demonstrated that inhibition of the Wnt signaling pathway can lead to inhibition of cell proliferation and cell cycle, as well as the expression levels of β-catenin, which are associated with the activation and shutdown of Wnt signaling that regulates expression levels of downstream target genes, such as cyclin D1, C-myc, E-cadherin and Lgr5, at the transcriptional level, thereby influencing the progression of tumors (21)(22)(23)(24)(25). ...
The present study aimed to investigate the effects of electrical stimulation (ES) on cell activity, cell cycle and apoptosis in injured rat dorsal root ganglion (DRG) cells induced by cyclic mechanical stretching (CMS). The present study also investigated whether the Wnt/β‑catenin pathway is involved in this process. Injury and ES models were established in DRG cells. Then, cell activity was detected using a Cell Counting Kit‑8 and 5‑ethynyl‑2'‑deoxyuridine‑594 cell proliferation assay kit. Cell cycle distribution was detected using a cell cycle detection kit. Apoptosis was detected using an Annexin V‑FITC apoptosis detection kit, and Wnt/β‑catenin pathway‑associated proteins were detected using western blotting. The present study demonstrated that CMS decreased DRG cell activity, increased the number of cells in the S phase, promoted cell apoptosis and inhibited the Wnt/β‑catenin pathway. In addition, ES significantly increased the proliferation activity of DRG cells, increased the number of cells in the G2 phase, decreased the apoptotic rate and activated the Wnt/β‑catenin pathway, ultimately reversing the injury caused by CMS. Following inhibition of the Wnt/β‑catenin signaling pathway using XAV939, the effects of ES were weakened. In conclusion, the present study demonstrated that ES may reverse CMS‑induced injury in DRG cells, and that the Wnt signaling pathway may be involved in this process.
... H630 cells and the 5-FU-resistant subclone H630R1 were equally sensitive to BOL. The higher IC 50 values may be related to the increased expression of ABCB1 in these cells [14], possibly resulting in greater efflux of the quassinoids. ...
STAT3, a transcriptional mediator of oncogenic signaling, is constitutively active in ~70% of human cancers. The development of STAT3 inhibitors remains an active area of research as no inhibitors have yet to be approved for the treatment of human cancer. Herein, we revealed that bruceantinol (BOL) is a novel STAT3 inhibitor demonstrating potent antitumor activity in in vitro and in vivo human colorectal cancer (CRC) models. BOL strongly inhibited STAT3 DNA-binding ability (IC50 = 2.4 pM), blocked the constitutive and IL-6-induced STAT3 activation in a dose- and time-dependent manner, and suppressed transcription of STAT3 target genes encoding anti-apoptosis factors (MCL-1, PTTG1, and survivin) and cell-cycle regulators (c-Myc). Structure–activity relationship studies demonstrated that the C15 side chain on BOL affected its ability to bind STAT3. Administration of 4 mg/kg BOL significantly inhibited CRC tumor xenografts [p < 0.001], but no effect was observed in a STAT3−/− tumor model. Additional studies showed that BOL effectively sensitized MEK inhibitors through repression of p-STAT3 and MCL-1 induction, known resistance mechanisms of MEK inhibition. Taken together, our findings suggest BOL is a novel therapeutic STAT3 inhibitor that can be used either alone or in combination with MEK inhibitors for the treatment of human CRC.
... the β-tubulin in the different CRCs cell lines on pillars (fig 4.10 and 4.11)[350][351][352][353] . On the other hand, e-cadherins are type of cell adhesion molecules (CAM) whose loss is known to promote metastasis of cancer cells. ...
This thesis deals with understanding behaviour of different cancer cell types on microstructured topography. We studied the behaviour of osteosarcoma cell line (SaOS-2) on confined micropillar structures and in particular their nuclear deformation. We analysed the role of the cytoskeleton, focal adhesions (FAs), nucleoskeleton (LINC and lamin A) and chromatin in SaOS-2 deformation on micropillar topography. Actomyosin and vimentin intermediate filament were shown to play a crucial role in orchestrating nuclear deformation. We found that FAs arrangement was mostly on side walls of pillars and that the LINC-cytoskeletal connection was essential for the nuclear deformation process but not lamin A. Employing chemo-topography modulations of pillars and a computational simulation model we demonstrated that the pulling down forces and not pushing down forces drive the cellular-nuclear deformation in osteosarcoma cells. We also studied the nuclear deformation of SaOS-2 on hydrogel micropillars with different stiffness and chemistry. We saw that cell morphology, actin organization and FAs behaviour was modulated by the substrate mechanics and chemistry. To explore the role of cancer origin, we examined the behaviour of various colon carcinomas on various micro-topographies and found that the epithelial origin cancers are less responsive to microscale topography compared to mesenchymal origin cancerous cells. However, their behaviour was affected on large pits which resembled the intestinal crypt and villi arrangement in terms of size.
... Cell cycle is driven by the sequential activation of cyclins and cyclin-dependent kinases in all eukaryotic cells. 32,33 Wnt/β-catenin signaling promotes cell cycle progression by upregulating target genes, such as c-Myc and Cyclin D1. 34,35 Our data indicated that RTHF might, therefore, regulate mitotic events by means of inhibiting Wnt/β-catenin pathway. ...
Background
Radix Tetrastigma hemsleyani flavone (RTHF) is extracted from a traditional Chinese medicinal herb T. hemsleyani, which is conventionally used as a folk medicine for its anti-inflammation activity and antiviral activity. In this study, the effects of RTHF on inhibiting malignant biological properties in colorectal cancer (CRC) were evaluated by conducting both in vitro and in vivo experiments, and the underlying mechanism was investigated.
Materials and methods
Cell Counting Kit-8, colony formation, and flow cytometry assays were performed to evaluate the proliferation of RTHF-treated colon tumor cells. Migration and invasion capacities were also tested by cell wound scratch assay and Transwell invasion assay. Moreover, the antitumor effects of RTHF on azoxymethane/dextran sulfate sodium-induced colitis-related CRC were investigated in C57BL/6 mice. In addition, Western blot and/or quantitative reverse transcription polymerase chain reaction analysis were used to evaluate the expressions of Lgr5, Cyclin D1, c-Myc, and E-cadherin.
Results
These experiments showed that RTHF could decrease the cell growth kinetics and clone-forming capacity. RTHF could also dose dependently induce cell cycle arrest at G0/G1 phase and inhibit epithelial-mesenchymal transition process. Furthermore, downregulation of β-catenin activation and downstream protein expression were detected in CRC cells after being treated with RTHF. RTHF daily gavage suppressed the number and size of CRC in mice and inhibited Lgr5 and Cyclin D1 expressions in tumor tissue.
Conclusion
In conclusion, RTHF treatment inhibits colorectal tumor growth, decreases Wnt/β-catenin pathway activity, and downregulates target genes’ expression.
... The development of resistance to chemotherapeutic agents remains a major challenge to chemotherapy to treat ileocecal adenocarcinoma. Chemotherapeutic agents such as vincristine sulphate, 5-fluorouracil, and (20). However, cancer cells become resistant when these chemotherapeutic agents are used for a prolonged period. ...
Multidrug resistance (MDR) represents a clinical obstacle to cancer chemotherapy since it causes cancer recurrence and metastasis. Acetyl-11-keto-β-boswellic acid (AKBA), an active ingredient derived from the plant Boswellia serrata, has been found to inhibit the growth of a wide variety of tumor cells, including glioma, colorectal cancer, leukemia, human melanoma, hepatocellular carcinoma, and prostate cancer cells. However, the actions of AKBA in multidrug-resistant cancer cells have not been fully elucidated. The current study examined the reversal of MDR by AKBA in a human ileocecal adenocarcinoma cell line with vincristine-induced resistance, HCT-8/VCR. A 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay indicated that cytotoxicity increased drastically and the IC50 of VCR in HCT-8/VCR cells decreased in the presence of AKBA. AKBA had a maximum "fold reversal" of MDR (FR) of 9.19-fold. In addition, HCT-8/VCR cells treated with AKBA and VCR exhibited a higher percentage of apoptotic tumor cells according to flow cytometry. The reversal of MDR by AKBA was evident in an intracellular increase in Rhodamine (Rh123), indicating that the activity of P-glycoprotein (P-gp) was blocked. Furthermore, AKBA inhibited the expression of P-gp and decreased levels of expression of multidrug resistance gene 1 in HCT-8/VCR cells. The current results indicated that AKBA might be a potential agent to reverse MDR in human ileocecal adenocarcinoma.
