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

The effects of MTC 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 info@gwyntwr1386.com

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).

HeLa is the oldest human cell line which is derived from cervical cancer cells taken on 1951, from Henrietta Lacks, a 31-year-old African-American (4).

Here we analyse the action of MTC in HeLa cells.

Time-course of MTC in Hela cells

In the present study our goal was to characterize the action of MTC on solid tumor cell lines such as HeLa. To this aim, HeLa cells were introduced with MTC (1μm) during different treatment hours. We found that HeLa cells started to be arrested in G2/M during the first 3-6 hours of treatment and they were completely arrested after 9, 18 and 24h of treatment. Apoptosis was detected at 24h treatment and cells were completely apoptotic by 48h treatment (Fig. 1).

Effects of MTC 10-6 M on cytoskeleton

To visualize the effects of 10-6 M MTC in microtubules in HeLa cells we stained the cells with Flutax-2 and DAPI. As it is shown in figure 2, microtubules were disrupted by MTC action at 12h treatment.

Western-blot analysis

Cyclins were analysed accordingly to different MTC incubation times. PARP and caspase-3, important for irreversible triggering of apoptosis, as well as JNK activity were also assayed.

We found that cyclin B1 expression increased till 24h and then decreased when apoptosis was triggered, expressions of cyclins D1and E raised until 12h and then decreased; cyclin A decreased continuously (Fig.3). PARP was broken down at 24 and 36h treatment corresponding to irreversible apoptosis triggering (Fig.3). C-Jun-GST increased continuously till 36h (Fig.3)

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).

Antibodies

C2.10 monoclonal anti-PARP, Enzyme Systems Products (Livermore, CA, USA); Anti-caspase 3 polyclonal rabbt (CPP32, YAMA, Apopain), Pharmigen international (San Diego, CA, USA); cyclin D1 (H-295): sc-753 rabbit polyclonal antibody, Santa Cruz Biotechnology (Santa Cruz, CA, USA); cyclin E (HE12): sc-247 mouse monoclonal antibody, Santa Cruz Biotechnology; cyclin B1 (H-432):sc752 rabbit polyclonal antibody, Santa Cruz Biotechnology; cyclin A (H-432):sc-751 rabbit polyclonal antibody, Santa Cruz Biotechnology; HRP Rabbit immunoglobulins, DAKO (Glostrup, Denmark); HRP Mouse immunoglobulins, DAKO; Anti-mouse IgG biotinylated whole antibody (fro goat), Amersham, Life Science (Buckinghamshire, UK); Anti-rabbit IgG biotinylated whole antibody (from Donkey), Amersham, Life Science; Streptavidin, horseradish peroxidase, Amersham, Life Science.

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

MTC (2-methoxy-5-92’,3’4’-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one). This compound was kindly given by Dr. JM Andreu and Dr. Thomas L. Fitzgerald (Florida A&M University, USA).

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. https://www.atcc.org/products/crm-ccl-2#:~:text=HeLa%20is%20an%20epithelial%20cell,transmitted%20disease%20research%2C%20and%20bioproduction.