Actin Cytoskeleton In Tumor Cells Research Articles

Chorein-dependent microfilament organization in tumor cells

Chorein is variably expressed in different cancer cells. In various non-malignant cell types, this protein regulates cytoskeleton microstructure and signaling. However, the role of chorein in regulating the actin cytoskeleton in tumor cells remains elusive. Here, we investigated chorein expression in various breast tumor cells and the involvement of this protein in microfilament organization. We used laser scanning microscopy to analyze microfilaments architecture, Triton X-100 fractionation to quantify G and Total actin levels, and quantitative RT-PCR to assess chorein gene transcription. We show that in line with previous observations, the less differentiated MCF7 breast cancer cells exhibited the highest relative expression of chorein compared to MDA-MB231 and T47D cell lines. Contrastingly, in less differentiated ZF rhabdomyosarcoma cells expressing high chorein levels, silencing of this protein was followed by clear depolymerization of actin microfilaments as apparent from IF morphological analysis. Quantification of G- and F-actin levels by Triton X-100 fractionation that revealed a significant increase in this ratio fully supported this finding. These results disclose that chorein is highly expressed in less differentiated tumor cells. In addition, silencing of this protein induces significant structural disorganization of the actin network, providing clear evidence that chorein regulates microfilament cytoskeleton architecture in tumor cells of higher malignant potential.

Macrocybin, a Natural Mushroom Triglyceride, Reduces Tumor Growth In Vitro and In Vivo through Caveolin-Mediated Interference with the Actin Cytoskeleton.

Mushrooms have been used for millennia as cancer remedies. Our goal was to screen several mushroom species from the rainforests of Costa Rica, looking for new antitumor molecules. Mushroom extracts were screened using two human cell lines: A549 (lung adenocarcinoma) and NL20 (immortalized normal lung epithelium). Extracts able to kill tumor cells while preserving non-tumor cells were considered “anticancer”. The mushroom with better properties was Macrocybe titans. Positive extracts were fractionated further and tested for biological activity on the cell lines. The chemical structure of the active compound was partially elucidated through nuclear magnetic resonance, mass spectrometry, and other ancillary techniques. Chemical analysis showed that the active molecule was a triglyceride containing oleic acid, palmitic acid, and a more complex fatty acid with two double bonds. The synthesis of all possible triglycerides and biological testing identified the natural compound, which was named Macrocybin. A xenograft study showed that Macrocybin significantly reduces A549 tumor growth. In addition, Macrocybin treatment resulted in the upregulation of Caveolin-1 expression and the disassembly of the actin cytoskeleton in tumor cells (but not in normal cells). In conclusion, we have shown that Macrocybin constitutes a new biologically active compound that may be taken into consideration for cancer treatment.

Abstract 6544: Macrocybin, a mushroom natural triglyceride, reduces tumor growth in vitro and in vivo through interference with the actin cytoskeleton

Abstract Mushrooms have been used for millennia in Eastern traditional medicine as cancer remedies and recent scientific studies have corroborated the presence of anticancer molecules in mushroom extracts. Our goal in this study was to screen a number of scarcely known mushroom species collected in the rain forest of Costa Rica looking for new antitumor molecules. Mushroom pieces were subjected to diverse extraction procedures involving water or ethanol at different temperatures. Extracts were screened using two human cell lines: A549 (lung adenocarcinoma) and NL20 (immortalized normal lung epithelium). Extracts able to kill tumor cells while preserving the integrity of non-tumor cells were considered “anticancer”. Positive extracts were fractionated with different techniques and the purification of the active compound was guided by its biological activity on the cell lines. When the compound was pure enough, its chemical structure was elucidated through nuclear magnetic resonance, mass spectrometry, and other ancillary techniques. The new molecule was synthesized and tested for biological activity both in vitro and in vivo with a xenograft assay. The mechanism of action was investigated through confocal microscopy, qRT-PCR, and Western blotting. The mushroom species with a better therapeutic window was Macrocybe titans, a local edible mushroom of very large proportions. The active ingredient was found in the precipitated phase of a 95% ethanol extraction with ultrasounds for 30 min, kept at -20°C for 24 h. To purify the active compound this extract was subjected to alkaloidal separation followed by column chromatography with polymeric resin (HP20-SS). The fraction obtained at pH 12 contained the active substance. Chemical analysis of the active molecule showed that it was a triglyceride containing oleic acid, palmitic acid, and a more complex fatty acid with 2 double bonds. Synthesis of all possible triglycerides with those characteristics and biological testing identified the natural compound as the R enantiomer of the triglyceride, which was named Macrocybin. A xenograft study showed that Macrocybin significantly reduces A549 tumor growth. In addition, Macrocybin treatment resulted in the dismantlement of the actin cytoskeleton in tumor cells (but not in normal cells). In conclusion, we have shown that the mushroom Macrocybe titans has anticancer properties and that the active principle is the new triglyceride, Macrocybin. This molecule constitutes a first-in-class new drug that works through the dismantlement of the actin cytoskeleton. Future experiments will establish the usefulness of Macrocybin for different types of cancer, either alone or in combination with other therapies. Citation Format: Marcos Vilariño, Josune García-Sanmartín, Laura Ochoa-Callejero, Rafael Peláez, Alfredo Martinez. Macrocybin, a mushroom natural triglyceride, reduces tumor growth in vitro and in vivo through interference with the actin cytoskeleton [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6544.

Ras Transformation Overrides a Proliferation Defect Induced by Tpm3.1 Knockout.

Extensive re-organisation of the actin cytoskeleton and changes in the expression of its binding proteins is a characteristic feature of cancer cells. Previously we have shown that the tropomyosin isoform Tpm3.1, an integral component of the actin cytoskeleton in tumor cells, is required for tumor cell survival. Our objective was to determine whether cancer cells devoid of Tpm3.1 would evade the tumorgenic effects induced by H-Ras transformation. The tropomyosin isoform (Tpm) expression profile of a range of cancer cell lines (21) demonstrates that Tpm3.1 is one of the most broadly expressed Tpm isoform. Consequently, the contribution of Tpm3.1 to the transformation process was functionally evaluated. Primary embryonic fibroblasts isolated from wild type (WT) and Tpm3.1 knockout (KO) mice were transduced with retroviral vectors expressing SV40 large T antigen and an oncogenic allele of the H-Ras gene, H-RasV12, to generate immortalized and transformed WT and KO MEFs respectively. We show that Tpm3.1 is required for growth factor-independent proliferation in the SV40 large T antigen immortalized MEFs, but this requirement is overcome by H-Ras transformation. Consistent with those findings, we found that Tpm3.1 was not required for anchorage independent growth or growth of H-Ras-driven tumors in a mouse model. Finally, we show that pERK and Importin 7 protein interactions are significantly decreased in the SV40 large T antigen immortalized KO MEFs but not in the H-Ras transformed KO cells, relative to control MEFs. The data demonstrate that H-Ras transformation overrides a requirement for Tpm3.1 in growth factor-independent proliferation of immortalized MEFs. We propose that in the SV40 large T antigen immortalized MEFs, Tpm3.1 is partly responsible for the efficient interaction between pERK and Imp7 resulting in cell proliferation, but this is overidden by Ras transformation.

Correction: A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells

Correction: A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells

A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

Fluorescent Aplyronine A: Intracellular Accumulation and Disassembly of Actin Cytoskeleton in Tumor Cells

Nonstick: Aplyronine A (ApA) is an antitumor and apoptogenic macrolide of marine origin. We prepared a fluorescent derivative of ApA that accumulated strongly throughout the cytoplasm and caused the rapid disassembly of the actin cytoskeleton in tumor cells. We also established that ApA caused malfunction of cell adhesion and dephosphorylation of focal adhesion kinase with apoptosis.

The favorable impact of PIK3CA mutations on survival: an analysis of 2587 patients with breast cancer

The phosphatidylinositol-3 kinase (PI3K) pathway regulates a number of cellular processes, including cell survival, cell growth, and cell cycle progression. Consequently, this pathway is commonly deregulated in cancer. In particular, mutations in the gene PIK3CA that encodes the p110α catalytic subunit of the PI3K enzymes result in cell proliferation and resistance to apoptosis in vitro and induce breast tumors in transgenic mice. These data underscore the role of this pathway during oncogenesis. Thus, an ongoing, large-scale effort is underway to develop clinically active drugs that target elements of the PI3K pathway. However, conflicting data suggest that gain-of-function PIK3CA mutations may be associated with either a favorable or a poor clinical outcome, compared with the wild-type PIK3CA gene. In the current study, we performed a systematic review of breast cancer clinical studies. Upon evaluation of 2587 breast cancer cases from 12 independent studies, we showed that patients with tumors harboring a PIK3CA mutation have a better clinical outcome than those with a wild-type PIK3CA gene. Importantly, this improved prognosis may pertain only to patients with mutations in the kinase domain of p110α and to postmenopausal women with estrogen receptor-positive breast cancer. We propose three potential explanations for this paradoxical observation. First, PIK3CA mutations may interfere with the metastasis process or may induce senescence, which results in a better outcome for patients with mutated tumors. Secondly, we speculate that PIK3CA mutations may increase early tumor diagnosis by modification of the actin cytoskeleton in tumor cells. Lastly, we propose that PIK3CA mutations may be a favorable predictive factor for response to hormonal therapy, giving a therapeutic advantage to these patients. Ultimately, an improved understanding of the clinical impact of PIK3CA mutations is critical for the development of optimally personalized therapeutics against breast cancer and other solid tumors. This effort will be important to prevent or explain therapeutic failures and select patients who are most likely to respond to new therapies that inhibit the PI3K pathway.

Hsp90 inhibition induces destabilization of actin cytoskeleton in tumor cells: functional significance of Hsp90 interaction with F–actin

Objective To examine the role of heat shock protein 90 (Hsp 90) in the maintenance of actin cytoskeleton in human neuroblastoma tumor cells. Methods Co-precipitation experiments were performed to examine Hsp 90 interaction with actin. Hsp 90 and actin interactions were evaluated by protein refolding and acto-myosin motility assays. 17-(Allylamino)-17-demethoxygeldanamycin (17AAG) induced actin-cytoskeleton re-organization was examined by laser scanning confocal microcopy. Results It was shown that inhibition of Hsp 90 by 17AAG accelerates detergent induced cell lysis of neuroblastoma tumor cells through destabilization of actin cytoskeleton. The in vitro co-precipitation experiments showed that functional but not mutant Hsp 90 binds with F-actin. Among biochemical modifications, phopshorylation and oligomerization enhanced Hsp 90 binding with F-actin. F-actin binding to Hsp 90 interfered with Hsp 90 chaperone activity in protein refolding assays, and Hsp 90 binding to F-actin interfered with actin motility on myosin coated flow cell. In the combination treatment, 17AAG irreversibly augmented the effect of cytochalasin D, an inhibitor of actin polymerization. Conclusions It can be concluded that Hsp 90 binds to F-actin in tumor cells and maintains the cellular integrity. The results display a novel element of Hsp 90 inhibition in destabilizing the actin cytoskeleton of tumor cells, therefore suggest that 17AAG combination with cytoskeletal disruptor may be effective in combating cancer.

In vitro pharmacological characterizations of the anti-angiogenic and anti-tumor cell migration properties mediated by microtubule-affecting drugs, with special emphasis on the organization of the actin cytoskeleton

The aim of the present work is to investigate whether microtubule-affecting drugs including vincristine, vinblastine, vindesine and vinorelbine are able to produce an anti-angiogenic effect at non-cytotoxic doses in the same way of taxol. The cytotoxic effects were determined by means of the colorimetric MTT assay, and the anti-angiogenic effects on HUVEC cells growing on Matrigel and forming capillary networks. Sixteen additional drugs (camptothecin, SN38, topothecan, adriamycin, daunomycin, etoposide, bleomycin, melphalan, mitomycin C, TNP-470, cisplatin, carboplatin, 5-fluorouracil, methotrexate, suramin and batimastat) were used as control in order to test the specificity of the microtubule-affecting drug effects. We also investigated by means of videomicroscopy whether microtubule-affecting drugs could produce anti-migratory effects at non-cytotoxic doses on tumor cells. Finally, we used computer-assisted fluorescence microscopy to characterize the influence of microtubule-affecting drugs on the polymerization/depolymerization dynamics of the actin cytoskeleton in tumor cells. Our results show that taxol, vincristine and vindesine behave similarly in their ability to reduce the capillary network formation by HUVEC cells cultured on Matrigel. These anti-angiogenic effects appear at non-cytotoxic concentrations. In contrast, vinblastine and vinorelbine produce apparent anti-angiogenic effects by direct cytotoxicity. Microtubule-affecting agents are also able to significantly reduce the level of migration of tumor cells at non-cytotoxic concentrations, some of these effects may occur via modifications to the actin cytoskeleton organization. Several types of microtubule-affecting agents could be used as anti-angiogenic agents by administering them at non-cytotoxic concentrations, and some microtubule-affecting agents abandoned in pharmacological assays could turn out to be potent anti-migratory drugs acting on tumor cells, though without being too cytotoxic.