En rykande het forskningsrapport

En forskningsrapport utförd av ungerska och tyska forskare i ett samarbete blev offentlig igår 26/9, och publicerades i det vetenskapliga organet

Man beskriver sin gärning enligt följande:
The Beilstein Journal of Organic Chemistry is an international, peer-reviewed, Open Access journal. It provides a unique platform for rapid publication without any charges (free for author and reader) – Platinum Open Access. The content is freely accessible 365 days a year to any user worldwide. Articles are available online immediately upon publication and are publicly archived in all major repositories. In addition, it provides a platform for publishing thematic issues (theme-based collections of articles) on topical issues in organic chemistry.
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The journal publishes high quality research and reviews in all areas of organic chemistry, including organic synthesis, organic reactions, natural product chemistry, structural investigations, supramolecular chemistry and chemical biology.

Det handlar om ett samarbete mellan 3 ungerska universitet: Semmelwis, Academy of Sciences, Institute of Chemistry och det tyska universitetet University of Applied Sciences Kaiserslautern.
Forskarna har genomfört en omfattande studie beträffande cancer och hur få en tumör att sluta växa, sprida sig och kanske även oskadliggöras (?). Forskningsrapporten är betitlad :

Comparative cell biological study of in vitro antitumor and antimetastatic activity on melanoma cells of GnRH-III-containing conjugates modified with short-chain fatty acids

Received 03 Mar 2018, Accepted 30 Aug 2018, Published 26 Sep 2018
 
Abstract
Background: Peptide hormone-based targeted tumor therapy is an approved strategy to selectively block the tumor growth and spreading. The gonadotropin-releasing hormone receptors (GnRH-R) overexpressed on different tumors (e.g., melanoma) could be utilized for drug-targeting by application of a GnRH analog as a carrier to deliver a covalently linked chemotherapeutic drug directly to the tumor cells. In this study our aim was (i) to analyze the effects of GnRH-drug conjugates on melanoma cell proliferation, adhesion and migration, (ii) to study the mechanisms of tumor cell responses, and (iii) to compare the activities of conjugates with the free drug.
Results: In the tested conjugates, daunorubicin (Dau) was coupled to ⁸Lys of GnRH-III (GnRH-III(Dau=Aoa)) or its derivatives modified with ⁴Lys acylated with short-chain fatty acids (acetyl group in [⁴Lys(Ac)]-GnRH-III(Dau=Aoa) and butyryl group in [⁴Lys(Bu)]-GnRH-III(Dau=Aoa)). The uptake of conjugates by A2058 melanoma model cells proved to be time dependent. Impedance-based proliferation measurements with xCELLigence SP system showed that all conjugates elicited irreversible tumor growth inhibitory effects mediated via a phosphoinositide 3-kinase-dependent signaling. GnRH-III(Dau=Aoa) and [⁴Lys(Ac)]-GnRH-III(Dau=Aoa) were shown to be blockers of the cell cycle in the G2/M phase, while [⁴Lys(Bu)]-GnRH-III(Dau=Aoa) rather induced apoptosis. In short-term, the melanoma cell adhesion was significantly increased by all the tested conjugates. The modification of the GnRH-III in position 4 was accompanied by an increased cellular uptake, higher cytotoxic and cell adhesion inducer activity. By studying the cell movement of A2058 cells with a holographic microscope, it was found that the migratory behavior of melanoma cells was increased by [⁴Lys(Ac)]-GnRH-III(Dau=Aoa), while the GnRH-III(Dau=Aoa) and [⁴Lys(Bu)]-GnRH-III(Dau=Aoa) decreased this activity.
Conclusion: Internalization and cytotoxicity of the conjugates showed that GnRH-III peptides could guard Dau to melanoma cells and promote antitumor activity. [⁴Lys(Bu)]-GnRH-III(Dau=Aoa) possessing the butyryl side chain acting as a “second drug” proved to be the best candidate for targeted tumor therapy due to its cytotoxicity and immobilizing effect on tumor cell spreading. The applicability of impedimetry and holographic phase imaging for characterizing cancer cell behavior and effects of targeted chemotherapeutics with small structural differences (e.g., length of the side chain in ⁴Lys) was also clearly suggested.
Keywords: drug-targeting conjugates; gonadotropin-releasing hormone-III; holographic microscopy; impedimetry; short-chain fatty acids

Ur den omfattande studien ( jag överdriver inte) klipper jag in det som är mest intressant för oss PHI,are.

 Holographic microscopic measurements were also performed to visualize the morphological changes induced by the conjugates and Dau. This novel technique provided several morphological parameters (e.g., surface area, optical thickness, eccentricity etc.) that allowed understanding of the results of impedance-based adhesion measurement.
Holographic images were taken before and after the 3-hour treatment of A2058 cells with the compounds. The change in the morphological parameters shown in Table S1 in Supporting Information File 2 was calculated from a fold change during 3 h long treatment and this value was normalized to that of the control cells. The adhesion inducer effects of Dau and GnRH-III(Dau=Aoa) detected by the impedimetric assay were reflected in the morphometry analysis. Both compounds could increase the surface area of adhered melanoma cells (124.1% ± 4.64, p < 0.001 and 115.7% ± 4.67, p < 0.05), while the optical thickness was reduced but only in case of the GnRH-III(Dau=Aoa) treated cells (85.6 ± 1.45, p < 0.001). Although [⁴Lys(Ac)]-GnRH-III(Dau=Aoa) proved to be an adhesion inducer, but it had a negative or no effect on these morphological indices (surface area: 84.2 ± 3.32 – 92.5 ± 3.5; thickness: 95.7 ± 1.94 – 103.1 ± 2.21). Opposite tendencies were shown for [⁴Lys(Bu)]-GnRH-III(Dau=Aoa), it could increase slightly, but significantly the surface area (114.0 ± 5.64, p < 0.05) and in parallel reduce the optical thickness (85.7 ± 1.85, p < 0.01) at 10⁻⁶ M concentration.

"Next, the chemokinetic activities (inducing random cell movement or locomotion) of conjugates were investigated by monitoring the locomotion of A2058 cells with holographic microscopy under the condition that the conjugates (10⁻⁷ to 10⁻⁵ M) were added directly to the cells in a uniform concentration. For the characterization of cellular movement, three parameters – migration (shortest distance), motility (actual path) and motility speed (ratio of actual path and time) were quantified by tracking single cells in time-lapse videos recorded by a HoloMonitor^TM M4 microscope (Phase Holographic Imaging AB, Lund, Schweden).
Based on the results, the GnRH-III(Dau=Aoa) and the ⁴Lys(Bu)-containing derivative conjugates displayed rather negative effects on the melanoma cell locomotion (Figure 7). By comparing the results of GnRH-III(Dau=Aoa) to the control this conjugate decreased the migration of A2058 cells in a concentration-dependent manner (Figure 7a), while a slight increase in the motility and the motility speed could be detected but only at 10⁻⁶ M concentration (Figure 7b and c). The highest concentration of [⁴Lys(Bu)]-GnRH-III(Dau=Aoa) decreased the migration, the motility and the motility speed of A2058 cells compared to that of the control cells (Figure 7g–i). However, the motility was slightly increased by 10⁻⁷ M [⁴Lys(Bu)]-GnRH-III(Dau=Aoa), the migration was similar to the control group (Figure 7g,h). These results could indicate that GnRH-III(Dau=Aoa) and [⁴Lys(Bu)]-GnRH-III(Dau=Aoa) would rather keep the cells in place. On the contrary, [⁴Lys(Ac)]-GnRH-III(Dau=Aoa) could increase all of the parameters at 10⁻⁷ M concentration (Figure 7d–f), which means the cells travel further in a more winding path with a higher velocity than the control cells. The migration inducer effect of 10⁻⁶ M [⁴Lys(Ac)]-GnRH-III(Dau=Aoa) indicated a more directed movement of cells (Figure 7d).

Figure 7: Effects of GnRH-III(Dau=Aoa) (a–c), [⁴Lys(Ac)]-GnRH-III(Dau=Aoa) (d–f) and [⁴Lys(Bu)]-GnRH-III(Dau=Aoa) (g–i) on the locomotion of A2058 cell line. The migration (a, d, g), motility (b, e, h) and motility speed (c, f, i) were investigated by HoloMonitor^TM M4 holographic microscopy. Dau: daunorubicin. Data shown in the figures represent averages calculated for 50 cell/group in 180 consecutive frames. The levels of significance are shown as follows *: p < 0.05; ***: p < 0.001.

Morphometry analysis
The experimental set up we used for the morphometry analysis can be also found in [6].
The HolomonitorTM M4 microscope (Phase Holographic Imaging AB, Lund, Sweden) was used for imaging and tracking the morphological changes of A2058 cells induced by the conjugates and Dau.
This technique is dedicated to analyzing the morphology and movement of adherent cells by recording 3D, holographic phase contrast images and tracking them over a given time period.
The principle behind this method is the detection of the phase shift of the probing laser light transmitting or reflecting through a cell sample and comparison to the reference light [7,8].
A2058 cells were seeded (2.5 × 105 cells) in a petri dish (diameter: 35 mm) and allowed to adhere for 24 h.
After automatic calibration of the background and microscope objective (20×), one field of each sample was focused on by the digital autofocus feature. The cells were then treated with the test compounds at the final concentrations of 10−7–10−5 mol/L, or with an equivalent volume of culture medium, and captured every 30 sec for 2 h.
For the evaluation, ca. 50 cells per time-lapse sequence were identified and selected by the minimum error histogram-based threshold algorithm of the HStudioTM M4 2.7.1 software (Phase Holographic Imaging AB, Lund, Sweden).
The morphological changes were automatically analyzed over time by selecting and tracking individual cells.
For the morphometry analysis, different basic and complex morphological parameters were displayed. The (i) area (surface area of the image occupied by a cell) and (ii) thickness (average spatial cell thickness) were grouped as basic metrics, while the more complex parameters relating to cell shape were: (iii) the eccentricity (how elongated a cell is comparing to a circle), (iv) the hull convexity (how different the 3D shape of a cell is compared to a perfect convex shape) and (v) the irregularity (how different the circumference of a cell is compared to the circumference of a perfect circle).

Study of cell movement
The movement A2058 cells induced by the conjugates and Dau (their chemokinetic effect) was investigated by HolomonitorTM M4 microscope.
Once the morphological changes of A2058 cells have been tracked, their cell movement was also be followed and registered over time.
For the characterization of the chemokinetic activity of the conjugates, the following
parameters were quantified: (i) migration (shortest distance between the starting point and the end point); (ii) motility (actual distance travelled by a cell from the starting point to the end point) and (iii) motility speed (ratio of actual path to time).
For the evaluation of the timelapse recordings, the same settings were used that in case of the morphometry study.

Min kommentar
Glasklart vad studien berättade och hur de använde sig av PHI`s HoloMonitor eller hur.
Inte? Nä knappast.Och självklart var den fylld med kemiska substanser och uttryck som är bloggens favoritområde (ironi). Men ok,jag ska ge mig på att försöka förstå och förklara vad den handlade om och hur viktig HoloMonitor`n var för dess slutsatser.
Idag när någon drabbas av cancer är det primära (om den upptäcks i tidigt stadie) att fort som f*n avlägsna tumören, ex via operation.Fakta är att de flesta dödsfall vid cancer inte sker av tumören i sig utan att den jäveln sprider sig, cancerceller från tumören sticker iväg uti kroppen, dvs metastaserar.
Väl ute i kroppen kan cellerna "gömma" sig och ligga inbäddade i vävnad ett bra tag för att plötsligt...
Dessa celler är oerhört svåra att hitta för läkarkåren,därav vill man få bort ursprungstumören rapido.
Forskare världen över försöker hitta sätt att leta upp dessa spridda killer cells, se ex "vårt" team GlycoImaging med sin forskning i det området,Biomarkörer.
Majoriteten av alla cancerdödsfall beror just på att man inte upptäckte tumören i tid,utan den hann metastasera till riktigt jobbiga ställen.Som ex skelett,hjärna,hjärta men även samtidigt till andra ställen i kroppen.
Då blir det svårt att få bort fanskapet utan att det/de livsviktiga organet tar skada och....
Ni har hört uttrycket : Operationen var lyckad, vi fick bort det vi skulle men tyvärr avled patienten ändå.
Men tillbaka till rapporten.
Man beskriver inledningsvis en idag vedertagen teknik att få tumören (om man hittat den i tidigt stadium) att stanna upp i tillväxt och täppa till möjlig metastasering från den.
Detta ämne/teknik, peptider med hormoner har man kikat lite närmare på då den är bevisat effektiv.
Men nu ur en annan synvinkel.Man har identifierat varför tekniken fungerar : kroppens, cancercellernas receptorer accepterar detta ämne och gör så att tumören som sagt stannar upp i utvecklingen och då gör den behandlingsbar operabel.
Nu studerade man istället receptorerna (mottagningsfunktion) vid en melanoma, hudcancer och tillsatte substans i receptorerna för att se hur dessa skulle svara vid insättning av ovanstående ämne.
"Daunorubicin (Dau) was coupled to Lys of GnRH-III (GnRH-III(Dau=Aoa)) or its derivatives modified with Lys acylated with short-chain fatty acids (acetyl group in [Lys(Ac)]-GnRH-III(Dau=Aoa) and butyryl group in [Lys(Bu)]-GnRH-III(Dau=Aoa))."
Här behövde man ha järnkoll på hur Melanomacellerna agerade under hela processen.
Beskrivet enl "melanoma cell proliferation, adhesion and migration".
Och där var HoloMonitor`n oerhört viktig då holotekniken är outstanding för att live kunna kolla cellers utveckling och rörelsemönster.
Utan PHI`s HoloMonitor hade man inte fått det resultat studien levererade.
Nämligen att en melanoma kan fås att "krympa" och i förlängningen oskadliggöras.
Eller som forskarna själva beskriver det : Internalization and cytotoxicity of the conjugates showed that GnRH-III peptides could guard Dau to melanoma cells and promote antitumor activity.
I samma slutsats får man även med hur viktig holotekniken var för resultatet : The applicability of impedimetry and holographic phase imaging for characterizing cancer cell behavior and effects of targeted chemotherapeutics with small structural differences (e.g., length of the side chain in Lys) was also clearly suggested.
Det framkommer redan i Abstractet då man tar med tekniken som en av de viktigaste beståndsdelarna i studien.
Keywords: drug-targeting conjugates; gonadotropin-releasing hormone-III; holographic microscopy; impedimetry; short-chain fatty acids

                                     Voila! Bloggen har uppfyllt sin del av dealen.

Ps. Ovanstående tycker bloggen är klart kursdrivande då forskarna så tydligt visar HoloMonitorn`s förträfflighet. Ds
Pps. Undertecknad är starkt haussigt inställd till Bolaget och dess potential vilket kan vara bra att veta.
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