The effects of MTC analogues in the cell cycle, cytoskeleton and apoptosis pathways in HeLa cells (ISSN 2753-8176 (online)

The effects of MTC analogues in the cell cycle, cytoskeleton and apoptosis pathways in HeLa cells

Ana Pedro 1

1 Gwyntwr1386 Pharmacy, Regus Chester Business Park, Heronsway, Chester, CH49QR, UK

MTC (2-Methoxy-5-(2’, 3’, 4’-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one) is a bicyclic colchine analogue synthesized by Fitzgerald (1) and induces microtubule disruption quickly and reversibly (2, 3) leading to a G2/M cell cycle arrest what culminates in apoptosis in human leukemic cells (Gajate et al, 2000, 4).

In order to unveil what MTC molecule parts or functional groups are important in G2/M arrest and apoptosis triggering, MTC analogues TKB, TCB, TMB were tested in different concentrations (Fig.1). MTC does nor have the B ring of colchicine (green arrow) and MTC analogues miss the carbonyl group of MTC (blue arrow).

Cell cycle analysis

We found that at 10-6, TCB and TMB did not exert any significant effect in HeLa cells, at 10-5 M, TMB arrested cells in G2/M after 9h treatment and after 36h treatment apoptosis was observed (Figs.2 and 3) and TCB arrested cells in G2/M after 12 h and after 36 h treatment they were already apoptotic. Also, we found that TKB at 10-6 M possessed the peculiar property of arresting cells in G2/M and then release them to return normal cell cycle (Fig.4). At 10-5 M, TKB was able to provoke G2/M arrest and apoptosis.

Cytoskeleton staining

We also examined HeLa cells treated with DAPI and Flutax staining in order to observe te action of these compounds on microtubules (Fig.5). TKB 10-6 arrests cells in G2/M (see the amount of mitosis in fig.5A, white arrow) and then releases cells, letting them go back to normal cycle (fig.5B). At 7h treatment, TCB and TMB 10-5 M had already quite disrupted the microtubules and we could visualize some mitosis (white arrows, Fig.5 C) what meant cells were arrested in G2/M.

Methods

This work was performed under the scope of a Masters thesis at University of Salamanca within the PhD program in Microbiology and Genetics (2002/2002).

Staining

DAPI (4’,6- diamino-2-phenylindole), Sigma

Flutax-2 (7-O-[N-(2,7-difluoro-4’-fluresceincarbonyl-L-alanyl] (with difluorofluorescein)- a kind gift of Drs AU Acuna and F Amat (CSIC, Madrid, Spain)

Culture media

Dulbecco’s MEM 25MM Hepes (DMEN), GibcoBRL, Life Technologies supplemented with 10% (v/v) heat-inactivated fetal calf (FCS), 2mM L-glutamine, 100 units/ml penicillin and 24 ug/ml gentamicin.

Cells

HeLa cells

Drugs

2,3,4-trimethoxy-4'-carbomethoxy-1,1'-biphenyl (TCB)

2,3,4-trimethoxy-4'-acetyl-1,1'-biphenyl (TKB)

2,3,4,4'-tetramethoxy-1,1'-biphenyl (TMB)

Instruments

- Citometry – Becton Dickinson – FACS calibu

- Confocal microscopy – Zeiss LSM 510

Programmes

- INPLOT-4 – protein concentration calculation

- Winmdi – flow citometry

- LSM 5 Image Browser – confocal

References

1.Fitzgerald TJ (1976). Molecular features of colchicine associated with antimitotic activity and inhibition of tubulin polymerization. Biochem Pharmacol1976 Jun 15;25(12):1383-7

2. Mollinedo F et al (1989). Cytoplasmic microtubules in human neutrophil degranulation: reversible inhibition by the colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1- one. Mol Pharmacol 36(4), 547-55

3. Gajate et al (2000). Induction of apoptosis in leukemic cells by the reversible microtubule-disrupting agent 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1 -one: protection by Bcl-2 and Bcl-X(L) and cell cycle arrest. Cancer Research 60, 2651-2659

4. Pedro A (2023). The effects of MTC in the cell cycle, cytoskeleton and apoptosis pathways in HeLa cells, ISSN 2753-8176 (online), DOI:10.13140/RG.2.2.21767.37283