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Ludmiła Węglarz 1Beata Parfiniewicz 1Arkadiusz Orchel 2Zofia Dzierżewicz 2

1. Medical University of Silesia, Department of Biochemistry, Narcyzow 1, Sosnowiec 41-200, Poland
2. Department of Biopharmacy, Medical University of Silesia, Narcyzów 1, Sosnowiec 41-200, Poland


Inositol hexaphosphate (IP6), a natural dietary ingredient has revealed anti-carcinogenic effect in various in vivo and in vitro models including colon cancer. Several studies have suggested that calcium antagonists, such as verapamil (VP) had inhibitory influence on cell proliferation or might potentiate the effects of numerous chemotherapeutic drugs on malignant cells. The purpose of this study was to evaluate the growth inhibitory activity of IP6 in combination with VP in comparison to that of IP6 alone and VP alone using two colon carcinoma-derived cell lines Caco-2 and HT-29. In combination treatment, IP6 (0.5, 1 and 5 mM) and VP (0.1, 0.5 mM) were added simultaneously and sequentially with VP added 2 h prior to IP6 to the cell cultures. The cells were cultivated for 72 h in RPMI 1640 medium at 37 °C in a humidified atmosphere with 5% CO2. Cellular growth was quantified with the use of CyQUANT Cell Proliferation Assay Kit based on cellular DNA content determination. Cell morphology following VP and IP6 treatment was evaluated using an Olympus IX 50 light microscope under 100x magnification. Histochemical assessment of nuclear morphological features associated with apoptosis/necrosis was performed on the basis of staining characteristics of VP-and IP6-treated cells with hematoxyline/eozine. Additionally, necrotic cells were characterized by trypan blue uptake. The activity of caspase-3, the chief executor of apoptosis, in colonic cells was estimated with the use of the Colorimetric Caspase-3 Assay Kit (Sigma) with Ac-DEVD-pNA as a substrate. The results of this study showed that VP alone reduced colon cancer growth and HT-29 cells appeared to be more susceptible to growth inhibition compared to Caco-2 cells. Statistically significant inhibition of Caco-2 cells proliferation was observed at VP doses ≥ 0.5 mM, whereas its growth inhibitory activity in regard to HT-29 cells was observed at 0.1 mM VP. The 1 mM VP appeared to be highly cytotoxic, resulting in a reduction of cell proliferation by 100-fold. IP6 at 5 mM induced significant growth suppression of both Caco-2 and HT-29 cells and its lower doses were ineffective. The joint administration of 0.5 mM VP and 1 mM IP6 enhanced growth inhibitory effect of IP6 compared to 1 mM IP6 alone on HT-29 but not Caco-2 cells. Substantial growth suppression of HT-29 cells was achieved when either 0.5 or 1 mM VP was administered prior to the addition of 1 and 5 mM IP6. Although some of nuclear changes associated with apoptosis such as chromatin condensation and nuclear fragmentation appeared sporadically after cells treatment with 0.5 and 1 mM VP, caspase-3 activation in cells of both lines was not observed following drug treatment, indicating that VP did not induce cell apoptotic death. The percentage of apoptotic cells under treatment with IP6 was dependent on its concentration, however, microscopic examination of trypan blue-stained cells treated with VP and IP6 revealed prevalence of morphological changes typical for necrosis.


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Presentation: Poster at V Multidyscyplinarna Konferencja Nauki o Leku, by Ludmiła Węglarz
See On-line Journal of V Multidyscyplinarna Konferencja Nauki o Leku

Submitted: 2006-01-30 13:51
Revised:   2009-06-07 00:44