Anoikis Resistance as a Further Trait of Acidic-Adapted Melanoma Cells

Melanoma is characterized by a low extracellular pH, which contributes to the development of an aggressive phenotype characterized by several properties as the switch to an epithelial-to-mesenchymal program, the increase of apoptotic resistance, and the migratory ability together with the development of drug resistance. Here, we demonstrate that melanoma cells grown in low pH medium (pH 6.7) for a short (24 hours) or long (at least 3 months) period equally express an anoikis resistance profile. Anoikis is a form of apoptosis prompted by loss of adhesion, particularly requested by aggressive cancer cells to metastasize. Anoikis resistance was ascertained in acidic melanoma cells either grown in agarose-coated plates or incubated in rocking conditions. Both analyses indicate that acidic cells were more able to survive in a nonadherent condition than cells grown in standard pH, an effect resulting in a more cloning efficiency and migratory ability. Ability to survive during rocking was inhibited using mTOR/NF-kB inhibitors. Finally, we checked whether characteristics related to thein vitroanoikis resistance acquired by acidic melanoma cells might be also suitable forin vivochallenge. We injected acidic melanoma cells into blood stream, and then we verify how many cells survived in blood after 15 min from the injection. Only acidic cells, transient and chronic, survived, whereas melanoma cells grown in standard pH medium did not. Overall, we have had the opportunity to demonstrate that low extracellular pH represents an additional mechanism able to promote an anoikis resistance in solid tumors.

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Nucleolar protein 7 facilitates melanoma progression and metastasis via HIF-1ɑ/PI3K/AKT/ERK axis

10.21203/rs.3.rs-86056/v1 ◽

2020 ◽

Author(s):

Yumei Li ◽

Fan Chen ◽

Weiyu Shen ◽

Bifei Li ◽

Ning Zheng ◽

...

Keyword(s):

Regulatory Networks ◽

Melanoma Cells ◽

Nucleolar Protein ◽

Functional Verification ◽

Loss Of Function ◽

Anoikis Resistance ◽

Nude Mouse Model ◽

Melanoma Progression

Abstract BackgroundMetastasis is the cause of most fatalities in cancer patients and is poorly understood. Discovery of the underlying determinant and regulatory networks implicated in the cancer cell metastasis is urgently needed. MethodsThe expression pattern of nucleolar protein 7 (NOL7) was examined by IHC in clinic melanoma samples. Loss-of-function in melanoma cells was achieved through siRNA and CRISPR/Cas9 system. Assays for proliferation, apoptosis and aggressiveness were performed for functional verification. Immunoblotting and qRT-PCR were used to measure the alterations in proteins and mRNA. Orthotopic xenograft nude mouse model was established to assess the effects of NOL7 on melanoma tumorigenicity and metastatic potential.ResultsNOL7 expression was increased with melanoma progression. Abrogation of NOL7 expression led to the dysfunction of malignant behaviors including proliferation, invasion, and anoikis-resistance of melanoma cells in vitro. Depletion of NOL7 expression suppressed melanoma growth and metastasis in vivo. Mechanistically, NOL7 was found to be induced by HIF-1ɑ under hypoxic condition and inhibition of NOL7 reduced the phosphorylation of AKT and ERK protein. ConclusionThis study revealed the cancer-promoting activity of NOL7 in melanoma cells and identified a novel regulatory mechanism of HIF-1ɑ/NOL7/PI3K/AKT/ERK axis in melanoma. NOL7 can function as a novel alert marker and therapeutic target for melanoma treatment.

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Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

Author(s):

Kathleen M. Donnelly ◽

Michael E. Bromberg ◽

Aaron Milstone ◽

Jennifer Madison McNiff ◽

Gordon Terwilliger ◽

...

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Factor Xa ◽

Coagulation Factor ◽

Melanoma Cells ◽

Factor V ◽

Ancylostoma Caninum ◽

Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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Treatment with Uncaria tomentosa Promotes Apoptosis in B16-BL6 Mouse Melanoma Cells and Inhibits the Growth of B16-BL6 Tumours

Molecules ◽

10.3390/molecules26041066 ◽

2021 ◽

Vol 26 (4) ◽

pp. 1066

Author(s):

Ali Zari ◽

Hajer Alfarteesh ◽

Carly Buckner ◽

Robert Lafrenie

Keyword(s):

Tumour Size ◽

Melanoma Cells ◽

Tunel Staining ◽

Aqueous Extracts ◽

Ki 67 ◽

Uncaria Tomentosa ◽

Mouse Melanoma ◽

Anti Cancer

Uncaria tomentosa is a medicinal plant native to Peru that has been traditionally used in the treatment of various inflammatory disorders. In this study, the effectiveness of U. tomentosa as an anti-cancer agent was assessed using the growth and survival of B16-BL6 mouse melanoma cells. B16-BL6 cell cultures treated with both ethanol and phosphate-buffered saline (PBS) extracts of U. tomentosa displayed up to 80% lower levels of growth and increased apoptosis compared to vehicle controls. Treatment with ethanolic extracts of Uncaria tomentosa were much more effective than treatment with aqueous extracts. U. tomentosa was also shown to inhibit B16-BL6 cell growth in C57/bl mice in vivo. Mice injected with both the ethanolic and aqueous extracts of U. tomentosa showed a 59 ± 13% decrease in B16-BL6 tumour weight and a 40 ± 9% decrease in tumour size. Histochemical analysis of the B16-BL6 tumours showed a strong reduction in the Ki-67 cell proliferation marker in U. tomentosa-treated mice and a small, but insignificant increase in terminal transferase dUTP nick labelling (TUNEL) staining. Furthermore, U. tomentosa extracts reduced angiogenic markers and reduced the infiltration of T cells into the tumours. Collectively, the results in this study concluded that U. tomentosa has potent anti-cancer activity that significantly inhibited cancer cells in vitro and in vivo.

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The phosphatase Shp1 interacts with and dephosphorylates cortactin to inhibit invadopodia function

Cell Communication and Signaling ◽

10.1186/s12964-021-00747-6 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Alessia Varone ◽

Chiara Amoruso ◽

Marcello Monti ◽

Manpreet Patheja ◽

Adelaide Greco ◽

...

Keyword(s):

Extracellular Matrix ◽

Tyrosine Phosphatase ◽

Melanoma Cells ◽

Matrix Degradation ◽

Extracellular Matrix Degradation ◽

Invadopodia Formation ◽

Interference Rna

Abstract Background Invadopodia are actin-based cell-membrane protrusions associated with the extracellular matrix degradation accompanying cancer invasion. The elucidation of the molecular mechanisms leading to invadopodia formation and activity is central for the prevention of tumor spreading and growth. Protein tyrosine kinases such as Src are known to regulate invadopodia assembly, little is however known on the role of protein tyrosine phosphatases in this process. Among these enzymes, we have selected the tyrosine phosphatase Shp1 to investigate its potential role in invadopodia assembly, due to its involvement in cancer development. Methods Co-immunoprecipitation and immunofluorescence studies were employed to identify novel substrate/s of Shp1AQ controlling invadopodia activity. The phosphorylation level of cortactin, the Shp1 substrate identified in this study, was assessed by immunoprecipitation, in vitro phosphatase and western blot assays. Short interference RNA and a catalytically-dead mutant of Shp1 expressed in A375MM melanoma cells were used to evaluate the role of the specific Shp1-mediated dephosphorylation of cortactin. The anti-invasive proprieties of glycerophosphoinositol, that directly binds and regulates Shp1, were investigated by extracellular matrix degradation assays and in vivo mouse model of metastasis. Results The data show that Shp1 was recruited to invadopodia and promoted the dephosphorylation of cortactin at tyrosine 421, leading to an attenuated capacity of melanoma cancer cells to degrade the extracellular matrix. Controls included the use of short interference RNA and catalytically-dead mutant that prevented the dephosphorylation of cortactin and hence the decrease the extracellular matrix degradation by melanoma cells. In addition, the phosphoinositide metabolite glycerophosphoinositol facilitated the localization of Shp1 at invadopodia hence promoting cortactin dephosphorylation. This impaired invadopodia function and tumor dissemination both in vitro and in an in vivo model of melanomas. Conclusion The main finding here reported is that cortactin is a specific substrate of the tyrosine phosphatase Shp1 and that its phosphorylation/dephosphorylation affects invadopodia formation and, as a consequence, the ability of melanoma cells to invade the extracellular matrix. Shp1 can thus be considered as a regulator of melanoma cell invasiveness and a potential target for antimetastatic drugs.

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Effect of cannulation site on emboli travel during cardiac surgery

Journal of Cardiothoracic Surgery ◽

10.1186/s13019-021-01564-1 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Mira Puthettu ◽

Stijn Vandenberghe ◽

Stefanos Demertzis

Keyword(s):

Cardiac Surgery ◽

In Vitro Study ◽

Blood Stream ◽

Air Bubbles ◽

Arterial Tree ◽

Air Bubble ◽

Cannulation Site ◽

The Brain

Abstract Background During cardiac surgery, micro-air emboli regularly enter the blood stream and can cause cognitive impairment or stroke. It is not clearly understood whether the most threatening air emboli are generated by the heart-lung machine (HLM) or by the blood-air contact when opening the heart. We performed an in vitro study to assess, for the two sources, air emboli distribution in the arterial tree, especially in the brain region, during cardiac surgery with different cannulation sites. Methods A model of the arterial tree was 3D printed and included in a hydraulic circuit, divided such that flow going to the brain was separated from the rest of the circuit. Air micro-emboli were injected either in the HLM (“ECC Bubbles”) or in the mock left ventricle (“Heart Bubbles”) to simulate the two sources. Emboli distribution was measured with an ultrasonic bubble counter. Five repetitions were performed for each combination of injection site and cannulation site, where air bubble counts and volumes were recorded. Air bubbles were separated in three categories based on size. Results For both injection sites, it was possible to identify statistically significant differences between cannulation sites. For ECC Bubbles, axillary cannulation led to a higher amount of air bubbles in the brain with medium-sized bubbles. For Heart Bubbles, aortic cannulation showed a significantly bigger embolic load in the brain with large bubbles. Conclusions These preliminary in vitro findings showed that air embolic load in the brain may be dependent on the cannulation site, which deserves further in vivo exploration.

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The extracellular matrix of hybrids between melanoma cells and normal fibroblasts

Journal of Cell Science ◽

10.1242/jcs.91.2.281 ◽

1988 ◽

Vol 91 (2) ◽

pp. 281-286

Author(s):

M.C. Copeman ◽

H. Harris

Keyword(s):

Extracellular Matrix ◽

Protease Activity ◽

Malignant Tumour ◽

Melanoma Cells ◽

Chromosome Loss ◽

Type I ◽

Chromosome 4 ◽

Hybrid Cells

It has been shown that when malignant tumour cells are fused with normal fibroblasts the suppression of malignancy in the hybrids is linked to their ability to produce a collagenous extracellular matrix in vivo. When, as a consequence of chromosome loss, segregants arise that reacquire malignancy, these do not produce any detectable matrix. In this paper we examine the main components of the extracellular matrix produced in vitro by hybrids between malignant mouse melanoma cells and normal mouse fibroblasts. Hybrids in which malignancy is suppressed synthesize about ten times as much type 1 procollagen as the malignant segregants derived from them; they also retain more fibronectin in the cell layer and release less protease activity into the medium. Malignant segregants more closely resemble the parental melanoma cells in producing fibronectin and mainly types IV and V procollagen. When hybrid cells in which malignancy is initially suppressed are grown continuously in vitro, the production of type I procollagen declines, and the production of type V procollagen and the release of protease activity into the medium increase. These changes, which are associated with the loss from the hybrid cells of both copies of the chromosome 4 derived from the parental fibroblast, predict the reacquisition of malignancy when the cells are inoculated into mice. It is possible that one gene or set of genes located on chromosome 4 determines both the execution of the fibroblast differentiation programme and the suppression of malignancy.

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Correction to: Daphnane diterpenes inhibit the metastatic potential of B16F10 murine melanoma cells in vitro and in vivo

BMC Cancer ◽

10.1186/s12885-018-4805-8 ◽

2018 ◽

Vol 18 (1) ◽

Author(s):

Myra O. Villareal ◽

Yuki Sato ◽

Kyoko Matsuyama ◽

Hiroko Isoda

Keyword(s):

Metastatic Potential ◽

Melanoma Cells ◽

Murine Melanoma ◽

Daphnane Diterpenes ◽

Murine Melanoma Cells

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Natural products with therapeutic potential in melanoma metastasis

Natural Product Reports ◽

10.1039/c4np00130c ◽

2015 ◽

Vol 32 (8) ◽

pp. 1170-1182 ◽

Cited By ~ 41

Author(s):

A. AlQathama ◽

J. M. Prieto

Keyword(s):

Natural Products ◽

Cancer Treatment ◽

Therapeutic Potential ◽

Melanoma Cells ◽

Mechanisms Of Action ◽

Melanoma Metastasis ◽

Pharmacological Effects ◽

Cytotoxic Compounds

Natural products continue to provide lead cytotoxic compounds for cancer treatment but less attention has been given to antimigratory compounds. We here systematically and critically survey more than 30 natural products with direct in vitro and in vivo pharmacological effects on migration and/or metastasis of melanoma cells and chart the mechanisms of action for this underexploited property.

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Intracellular pH-temperature interactions of hepatocytes from American eels

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1983.245.1.r32 ◽

1983 ◽

Vol 245 (1) ◽

pp. R32-R37

Author(s):

P. J. Walsh ◽

T. W. Moon

Keyword(s):

Intracellular Ph ◽

Acclimation Temperature ◽

C Cells ◽

Medium Ph ◽

American Eel ◽

Temperature Changes ◽

American Eels ◽

Temperature Interactions

The effects of acclimation temperature and acute temperature changes on the intracellular pH (pHi) of hepatocytes isolated from the American eel, Anguilla rostrata, were studied by the measurement of the distribution ratio of dimethyloxizolidinedione (DMO). Varying the concentration of DMO (10(-7) to 10(-4) M) did not affect estimates of pHi, indicating that DMO acts as an ideal pHi probe in eel hepatocytes. In vitro studies yielded values of liver cell pHi identical to those previously measured in vivo (in vitro pHi = 7.556 +/- 0.010; in vivo pHi = 7.570 +/- 0.049 at 20 degrees C); hepatocyte pHi varied inversely with acclimation temperature (5-20 degrees C) in a manner consistent with alphastat regulation (delta pH/delta T = -0.0182 +/- 0.021). During acute temperature increases (5-20 degrees C) and decreases (20-5 degrees C) hepatocytes regulated pHi to the appropriate (acclimated) value within 30-45 min posttransfer under conditions of constant medium pH (pHe). The effects of medium pH were also studied, and although patterns of pHi regulation differed between 5 and 20 degrees C cells, a pHi difference consistent with alphastat regulation was maintained between 5 and 20 degrees C cells over the pHe range 7.8-8.3.

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